Western Blotting: A Guide To Current Methods

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TA BLE O F C O N T E N T S
Western Introductions
Blotting: 2 Western blotting: Surveying the past to the present
A Guide

Tianna Hicklin, Ph.D.
Science/AAAS
to Current 3 A new standard in reproducible quantitative
Methods

Western blotting
Kim Cushing
Product Marketing Manager
GE Healthcare
Western blotting overview

4 A history of the old West(ern blot)
Jeffrey M. Perkel
7 Preparing for your Western blot

Jeffrey M. Perkel
10 Choosing your detection method

Jeffrey M. Perkel
Research articles
12 An analysis of critical factors for quantitative
immunoblotting
Kevin A. Janes
22 A neuronal antigen in the brains of Alzheimer patients

Benjamin L. Wolozin, Alex Pruchnicki, Dennis W. Dickson et al.
25 Serological analysis of a subgroup of human

About the cover: Western blotting images
T-lymphotropic retroviruses (HTLV-III) associated
provided courtesy of GE Healthcare. with AIDS
This booklet was produced by the Science/ Jörg Schϋpbach, Mikulas Popovic, Raymond V. Gilden et al.
AAAS Custom Publishing Office and
sponsored by GE Healthcare.
28 Cell surface P-glycoprotein associated with multidrug
Editor: Tianna Hicklin, Ph.D. resistance in mammalian cell lines
Proofreader/Copyeditor: Yuse Lajiminmuhip
Designer: Amy Hardcastle Norbert Kartner, John R. Riordan, Victor Ling
BILL MORAN, GLOBAL DIRECTOR

Custom Publishing
bmoran@aaas.org
+1-202-326-6438
Technical notes
ROGER GONCALVES, SALES MANAGER
31
Custom Publishing
Europe, Middle East, and India Reproducibility using the Amersham™ WB system
rgoncalves@science-int.co.uk
+41 43 243-1358
Åsa Hagner McWhirter, Anita Larsson, Elisabeth Wallby et al.
© 2015 by The American Association for the Advancement

of Science. All rights reserved. 9 June 2015
1
W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S
W
estern blotting—easily recognizable by the distinct
images of laddered bands that it generates—has
remained a relatively popular protein detection tech-
nique over the past 36 years.
From immunology to neuroscience to drug discovery, the fields to
which Western blots have contributed are wide ranging. Science and
its family of journals have published hundreds of research articles that
have used the technique since it was first described, as it has been
a useful tool in many scientific discoveries. Shortly after the method
was first described in 1979, for example, Western blots began being
Western used to unravel questions about which proteins were contributing to
drug resistance (see page 28) and Alzheimer’s disease (see page 22)
blotting: as well as to characterize the human retrovirus that was causing a new
epidemic, AIDS (see page 25).
Surveying Though it has remained a staple for protein detection over the
years, the technique is not immune from the growing concerns of
the past to the scientific community for increased reproducibility of data. Such
concerns have been recently described in an editorial by Science
the present Journals Editor-in-Chief Marcia McNutt in which she discussed the
importance of reproducibility, and transparency, when publishing
data (www.sciencemag.org/content/346/6210/679.full) as well as the
efforts a group of editors put forth to design guidelines for reporting
data in the hopes that they will be viewed “as part of the quality con-
trol that justifies the public trust in science.”
For Western blots in particular, the optimization of various steps
and a great deal of experience working with the technique are key to
gathering quality data that is reliable and reproducible. As a multi-
step process, the technique inherently houses many places in which
variability can be introduced during data collection. In a 2015 Sci-
ence Signaling article, Kevin A. Janes details some of the critical fac-
tors for achieving accurate immunoblotting data, including sample
preparation, loading controls, and choice of reagents and buffers
(see page 12).
In this booklet, we invite you to explore some of the history of
Western blotting as we take a look back at some of the highly cited
advances to which the method has contributed and explore some of
the many ways the technique can be optimized to help create higher
quality and more reproducible data.
Tianna Hicklin, Ph.D.

Editor, Science/AAAS Custom Publishing Office
2 sciencemag.org SCIENCE
SE C T I O N O N E | I N T RO D U C T IO N S
W
estern blotting, also known as immunoblotting when
first described in 1979, is today a ubiquitous method
in the life science laboratory. From cell biology to
protein purification and characterization, Western
blotting remains an essential protein analytical technique that is fun-
damental to protein research.
One of the biggest challenges for Western blotting is data
reproducibility. Within the multistep immunoblotting process, there
are many potential ways to introduce error and variation. This inherent
A new risk often necessitates that experiments are repeated many times in
order to generate reproducible data. Every step of the process has
standard in an impact on the result and must be controlled to ensure the quality
of the final data. This is particularly critical when data publication is
reproducible the ultimate goal and reproducible quantitative data is an absolute

prerequisite.
In 1990, Amersham International (now part of GE Healthcare) was
quantitative the first company to introduce enhanced chemiluminescent (ECL)
Western blotting detection reagents. Chemiluminescence is a highly
Western sensitive detection methodology and was the leading detection
technology used in Western blotting for many years; however, as the
blotting critical horseradish peroxidase enzyme reaction is dynamic, the light
produced declines over time making it challenging to produce truly
quantitative data.
Advances in charge-coupled device (CCD) technology have
led to the use of digital imaging systems that can capture both
chemiluminescent and fluorescent signals. When using fluorescence
detection, the signal generated by dye excitation is stable for several
months, producing consistent and quantitative results, making it an
excellent choice for true protein quantitation.
Since the introduction of the first ECL detection reagent for
Western blotting, the portfolio of products offered by GE Healthcare
has been expanded, improved, and optimized to cover the breadth of
Western blotting requirements from sensitive protein identification to
reproducible protein quantification. The most recent addition to the
range is the new Amersham WB system, which integrates separation,
transfer, detection, and quantitative analysis of proteins. This fully
integrated system minimizes assay variability to provide consistent,
quantitative Western blot data for every sample, every time.
Western blotting continues to be an essential technique which,
while undergoing significant improvements remains largely faithful to
the tenet of the original protocol. There are opportunities to optimize
and refine the Western blotting protocol that can deliver some real
benefits
In addition to this publication we were delighted to sponsor two
webinars, which highlight some of the key considerations for optimal
Western blotting:
Quantitative Western blotting:

Improving your data quality and reproducibility
Western blot tips and tricks:

Filling the gap between art and science
Kim Cushing
Product Marketing Manager
GE Healthcare
SCIENCE sciencemag.org 3
A history of the old

gel, are transferred to a membrane such that their pattern in the
gel is retained, like making a photocopy, or what Towbin called
“a faithful replica” (1). That “blot” is then probed using “primary”
West(ern blot) antibodies to a specific protein or proteins of interest, and de-

veloped by addition of secondary antibodies, which recognize
the primaries. These secondary antibodies are labeled with
By Jeffrey M. Perkel either a radioisotope, fluorophore, or enzyme, all of which allow
the proteins’ positions to be identified.
The method is largely unchanged—at least in broad strokes—
since Towbin, with Theophil Staehelin and Julian Gordon, used
it to detect bacterial or chicken ribosomal proteins that had
been separated on urea-containing polyacrylamide gels. They
W
hen Harry Towbin and colleagues at the Fried- detailed their method in the Proceedings of the National Acad-
rich Miescher Institute in Basel, Switzerland first emy of Sciences in 1979, proposing such applications as screen-
detailed the procedure that would come to be ing for monoclonal antibody-producing hybridomas, autoim-
called the Western blot, almost nothing was easy. mune sera, enzymatic activities, and ligand binding (1).
Antibodies weren’t readily available in 1979, so they made their Yet Towbin’s original article never actually names the meth-
own by injecting purified ribosomal proteins into mice or goats. od; it simply refers to an “electrophoretic blotting technique.”
Their transfer procedure involved a gel and nitrocellulose mem- The term “Western blot” was actually coined 2 years later by
brane sandwiched between two Scotch-Brite scouring pads W. Neal Burnette, of the Fred Hutchinson Cancer Research
and a “disposable micropipette tray” for structural support, and Center in Seattle, Washington, who described several technical
bound together with rubber bands. The transfer itself was con- improvements to the method, including applying it to the more
ducted in “an electrophoretic destaining chamber” (1). commonly used SDS-PAGE gels:
Secondary antibodies were a little easier, at least for fluores-
cent and colorimetric detection since these could be purchased. “With due respect to [Edwin] Southern, the established
But for radioisotopic detection, the researchers once again were tradition of ‘geographic’ naming of transfer techniques
on their own, labeling their own antibody preparations with (‘Southern,’ ‘Northern’) is continued; the method
iodine-125 via “the chloramine T method”—and exposing the la- described in this manuscript is referred to as ‘Western’
beled blots to film for 6 days. blotting” (2).
Today, the method is considerably easier: researchers can
purchase precast gels, transfer proteins to membranes using Unraveling multidrug resistance
commercial transfer apparatuses, and capture their data in One of the earliest applications of Towbin’s procedure used
seconds using digital imagers. Towbin’s landmark paper has the method to tease apart the mechanism of multidrug resis-
been cited more than 51,000 times in the intervening 36 years, tance in immortalized cells.
according to Google Scholar. Yet for the most part, the method Researchers had observed that while many cultured cells
remains largely the same. Modern Western blot sandwich were sensitive to drugs like colchicine and actinomycin D, some
cassettes bear a strong family resemblance to the one Towbin were resistant, and if they were resistant to one compound, they
dreamed up. And researchers still use fluorophore and enzyme- often were resistant to many. Molecular analysis determined
conjugated secondary antibodies to identify bands of interest, that a 170 kD membrane protein seemed to correlate with mul-
though most researchers now favor chemiluminescence over tidrug resistance, but it was not yet clear if that protein was ac-
colorimetric detection. tually causative of the phenotype, or merely a passenger.
That’s not to say methods developers have been content to In 1982, Victor Ling of the Ontario Cancer Institute and
rest on their laurels. Researchers have extended and evolved University of Toronto, and colleagues demonstrated that DNA
the method over the decades, making it easier, faster, and more transfer from drug-resistant cells to sensitive cells induced
reliable. a resistant phenotype, including the appearance of that 170
Here, we travel back to the Old Western, to see where the ven- kD protein on polyacrylamide gel separations of membrane
erable immunoblot has been and explore how far the technique protein preparations. But the band was diffuse and partially
has come. obscured by the complex membrane material, so, “To increase
the sensitivity and specificity of detecting the P-glycoprotein,”
The Western blot they wrote, “we employed an antiserum raised against CHO
A Western blot is simply a way to identify proteins on a poly- [Chinese hamster ovary] cell mutant membranes in a replica
acrylamide gel. Proteins, separated by size on a polyacrylamide Western blot procedure” (3).
SE C T I O N T W O | WESTERN BLOTTING OVERVIEW
Ling’s team didn’t To find out, Robert

use that antiserum Gallo and his team at
directly, however; the National Cancer
they cleaned it up first Institute in Bethesda,
by preabsorbing it Maryland, performed
against a preparation a battery of tests,
from drug-sensitive including electron
cells. In this way, microscopy, reverse
they were able to transcriptase enzyme
clearly detect a band assays, cell co-culture
consistent with the experiments—and
P-glycoprotein in drug- Western blots. In one
resistant but not sensi- series of experiments,
tive lines. the team infected
But those results, human T cells with
the researchers ac- the virus that they
knowledged, were still called HTLV-III, then
only correlative—they separated cellular
had no way of know- proteins (including the
ing, for instance, whether the P-glycoprotein gene was trans- virus) on protein gels and blotted them to nitrocellulose. They
ferred with other sequences that actually conferred the drug then cut those blots into strips and “tested [them] with samples
resistance itself. Much of that doubt, though, was eliminated of human serum in a strip radioimmunoassay (RIA) based on the
when the researchers demonstrated in 1983, again via Western Western blot technique” (5; see page 25). Those sera samples
blotting, that drug resistant cells from Chinese hamster, Syrian had been taken “from patients with AIDS or pre-AIDS, from
hamster, mouse, and human all contained the 170 kD P-glyco- contacts of such patients, and from homo- or heterosexual male
protein, all of which could be detected using antibodies raised controls.”
against membrane preparations from both drug-resistant CHO The goal was to determine whether blood samples collected
and human cell lines (4; see page 28). from patients with AIDS contained antibodies capable of
These data, of course, suggested that whatever the recognizing HTLV-III proteins, and as it turns out, they did.
P-glycoprotein is, it is evolutionarily conserved. But the Sera from patients with AIDS or pre-AIDS lit up specific bands
implications were broader than that. “Our findings could in virus-infected cellular lysates, but not uninfected control
have important implications for cancer therapy,” Ling wrote in cell lines. Control patient sera did not bind HTLV-III proteins.
Science. “It is possible that clinical resistance to combination The analysis also demonstrated that HTLV-III is distinct from,
chemotherapy might result from an unchecked proliferation though related to, other HTLV family members, and began
of tumor cell subpopulations with a multidrug resistance the painstaking process of teasing apart the virus’ molecular
phenotype” (4). composition (5).
Today, that prediction has been shown to be true. The In total, the Gallo team published four studies in the 4 May
170 kD P-glycoprotein is an ATP-binding cassette (ABC) 1984 issue of Science. In the final one, the team determined that
transporter that actively pumps drugs and other compounds blood sera from 43 of 49 AIDS patients and 11 of 14 pre-AIDS
out of cells, conferring drug resistance to cancer cells—a patients recognize antigens—particularly the 60 kDa env protein—
clinically important observation that was made possible via the found in a viral extract from HTLV-III-producing cells, in this case
PHOTO: © POLAKPHOTO/SHUTTERSTOCK.COM
Western blot. using ELISA and RIA based on the Western protocol (6).
“The data presented here and in the accompanying reports
Decoding AIDS suggest that HTLV-III is the primary cause of AIDS,” Gallo and
Another landmark in Western history occurred in 1984. At his team concluded (6). Within a few years, HTLV-III would be
the time, acquired immunodeficiency syndrome (AIDS) was rebranded with the name by which it is known today: human im-
a disease of unknown etiology that was impacting mostly munodeficiency virus (HIV).
intravenous drug users and homosexual men. The disease had That discovery, wrote Jean Marx in a news story accompany-
only been recognized in 1981, but within 2 years, researchers ing the reports, opened the door to the development of reagents
had determined that the causative agent likely was a retrovirus, for disease diagnostics and blood donor testing, as well as for re-
a relative of the human T-lymphotropic viruses (HTLV). The search into potential vaccines (7). And Western blotting played
question was: which one? a key role. continued>
W E S T E RN BLOT T ING : A G U I D E TO CU
M ORDREERNNTM
MEETH
THOODDSS
Untangling Alzheimer’s disease recycling. They then combined those figures with electron mi-
In 1986, researchers used Western blotting to address yet croscopy, mass spectrometry, and superresolution imaging data
another clinical puzzle, Alzheimer’s disease. to generate “a three-dimensional model of an ‘average’ synapse,
In an effort to begin to understand the biochemistry of dis- displaying 300,000 proteins in atomic detail” (12).
ease, Benjamin Wolozin and colleagues at the Albert Einstein As its applications have evolved, so too has the Western blot-
College of Medicine used brain extracts from control patients ting method itself. Shortly after the original Towbin paper, for
and patients with Alzheimer’s to identify an antibody that could instance, researchers developed the so-called Southwestern
distinguish diseased and non-diseased brain homogenates. blot (13). Literally a cross between Southern and Western blot-
The resulting reagent, called “Alz-50” bound an antigen that ting, the technique probes protein blots with labeled DNA to
was some 15 to 30 times more abundant in the temporal cortex, identify DNA-binding proteins.
hippocampus, and nucleus basalis of patients with Alzheimer’s Another variation is the “In-Cell Western,” combining the
disease than non-disease individuals (8; see page 22). Alz-50 features of Western blotting and ELISA. In a traditional ELISA,
stained neurons in the brains of patients with Alzheimer’s but an antibody to the target of interest is bound to a solid surface,
not control individuals, and also lit up the neurofibrillary tangles mixed with a sample, and then probed with a second, labeled
that characterize the disease. antibody. In an In-Cell Western, cells grown on the surfaces of
microtiter plates are lysed in situ, probed with primary antibod-
ies to the antigen(s) of interest, and finally detected using fluo-
rescently labeled secondary antibodies. The method avoids the
Researchers have extended and evolved need for gel electrophoresis and blotting, and thus is faster than
a traditional Western. But it also assumes no background bind-
the method over the decades, making it ing, as there is no electrophoretic separation step.
Methods developers have also found ways to accelerate and
easier, faster, and more reliable. optimize the Western procedure. As detailed in a Science maga-
zine technology feature, for instance, EMD Millipore’s SNAP i.d.
The question was, to what antigen was Alz-50 binding. Wolozin 2.0 system uses a vacuum manifold to reduce Western blotting
and colleagues enriched the antigen from Alzheimer and control incubation and washing steps “from 4 hours to 30 minutes” (9).
brain homogenates, resolved them on an SDS-polyacryl am- Others have developed methods to reduce blotting time and
ide gel, transferred to nitrocellulose, and probed the blot with increase throughput, such as ProteinSimple’s Simple Western
Alz-50. Detection, using an alkaline phosphatase-conjugated system. Simple Westerns replace the traditional SDS-PAGE
secondary antibody, revealed a 68 kD protein in diseased brain gel and blotting steps with capillary electrophoresis, which
homogenates that was absent in normal tissue (8). can separate proteins by size or charge. Up to 96 samples can
“Our results demonstrate that Alz-50 recognizes a protein automatically be run in parallel and detected in the capillaries
present in neuronal terminals in plaques and in most neurons directly via chemiluminescent detection. The resulting data are
with tangles,” Wolozin et al. concluded. But they had no idea then rendered as a traditional blot using the system software.
what that protein might be, though they doubted it could Another Western blotting optimization that has emerged
be tau protein, one of the few proteins known to be of the since Towbin is multiplexing. Researchers using fluorophore-
right size, as it did not exhibit the differential abundance they conjugated secondary antibodies now can detect two or more
observed. Today, researchers know that presumption was in- antigens simultaneously, which is particularly useful for quan-
correct: Alz-50 binds a phosphorylated form of tau, a major titation and normalization of protein abundance (see Prepar-
component of neurofibrillary tangles in so-called tauopathies, ing for your Western blot, page 7). In one typical example, the
including Alzheimer’s. antigen of interest is detected using one fluorescent color and a
housekeeping protein with a second. Because both proteins are
Technical innovations detected on a single gel and simultaneously, the method saves
Western blotting has since participated in studies across time. But it also improves reproducibility relative to the alterna-
the biological spectrum, from identifying the substrates of the tives, which are either to probe, strip, and reprobe the blot with
aging-associated Sir2 deacetylase and probing the biology of a second primary antibody, or to run duplicate gels and probe
synthetic prions to modeling neurotransmitter synaptic vesicle them with separate antibodies.
trafficking (10–12). Perhaps the most significant technical advances in Western
In the latter study, Silvio Rizzoli of the University of Göttingen blotting history, however, have been the most pedestrian. For
Medical Center, and colleagues, used “quantitative immunob- one thing, unless they are studying a new protein, researchers
lotting” to measure the abundance of each of 62 proteins in the rarely need, as Towbin did, to prepare their own antibodies;
so-called synaptosome, a structure involved in synaptic vesicle with some 1.8 million antibodies listed in Antibodypedia, it’s a
SE C T I O N T W O | WESTERN N O N E | EDITORIAL
SE C T I OBLOTTING OVERVIEW
Preparing for your

good bet somebody else has already done so. Meanwhile, us-
ing digital gel-documentation systems researchers can ditch
their manual cameras and lightrooms and capture digital data
instead. Such systems certainly are easier to use than X-ray film,
the traditional method of chemiluminescent detection, but they Western blot
also represent a tremendous advance in that they are more
sensitive, produce a linear signal over a wider dynamic range, By Jeffrey M. Perkel
and enable more reproducible experiments. Equally significant
is the development of methods to make what effectively is a
qualitative technique, quantitative—a development that pow-
ered the synaptosome modeling study, for instance (12, 14; see
page 12).
W
The remarkable thing is, for all these advances, the original estern blotting is ubiquitous in modern molecu-
Towbin protocol remains apparent. Countless labs around the lar biology laboratories. It’s relatively simple to
world still use plastic Western blotting cassettes and sponges perform, but good-looking data—and especially
that look remarkably like the sketch in the seminal 1979 paper. quantitative and reproducible data—can be hard to
The process is more efficient and straightforward than ever, come by. A complete Western blotting protocol comprises doz-
yet the fundamental principle of creating and probing a ens of steps, dozens of places in which something can go wrong,
“faithful replica” of a protein gel remains. As the Western blot from sample preparation all the way through to detection. Mak-
closes in on four decades, it will be interesting to see where it ing a protocol that is reproducible from day to day and lab to lab
goes next. such that researchers can be confident of anything but the most
dramatic differences in abundance can be a challenge. Here we
Jeffrey M. Perkel is a freelance science writer based in Pocatello, review some key steps to consider.
Idaho.
Sample preparation
References The first step in a Western blotting protocol is preparing a
1. H. Towbin et al., Proc. Natl. Acad. Sci. U.S.A. 76, 4350 cell or tissue lysate. The cell membranes must be broken open
(1979). to release the cellular contents, using a lysis or extraction buffer,
2. W. N. Burnette, Anal Biochem. 112, 195 (1981). and the protein(s) of interest solubilized so they can be sepa-
3. P. G. Debenham et al., Mol. Cell Biol. 2, 881 (1982). rated with gel electrophoresis. But there is no one best way to do
4. N. Kartner et al., Science 221, 1285 (1983). that. Some samples, for instance, require harsher lysis conditions
5. J. Schüpbach et al., Science 224, 503 (1984). than others, and not all proteins are soluble or stable in the same
6. M. G. Sarngadharan et al., Science 224, 506 (1984). detergent.
7. J. L. Marx, Science 224, 475 (1984). Lysis buffers “differ in their ability to solubilize proteins, with
8. B. L. Wolozin et al., Science 232, 648 (1986). those containing sodium dodecyl sulfate [SDS] and other ionic
9. A. Harding, Science, (2013), doi:10.1126/science.opms. detergents considered to be the harshest and therefore most
p1300073. likely to give the highest yield,” according to a guide published
10. V. J. Starai et al., Science 298, 2390 (2002). by antibody-developer Abcam (1). Some common nonionic
11. G. Legname et al., Science 305, 673 (2004). detergents include Triton X-100, Nonidet P-40, and Tween; ionic
12. B. G. Wilhelm et al., Science 344, 1023 (2014). detergents include SDS, deoxycholate, and hexadecyltrimethyl-
13. F. K. Y. Siu et al., Nat. Protocols 3, 51 (2008). ammonium bromide (CTAB).
14. K. A. Janes, Science Signaling 8, rs2 (2015). In a recent report in Science Signaling, Kevin Janes of the Uni-
versity of Virginia documented several “critical factors for quan-
titative immunoblotting,” including sample preparation (2; see
page 12). He noted, for instance, that the commonly used RIPA
buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100,
0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, plus protein-
ase and phosphatase inhibitors)—efficiently solubilizes cellular
proteins, though some are left behind in an insoluble pellet.
In Janes’ own hands, analysis of the soluble and insoluble
fractions resulting from RIPA lysis of HT-29 human colon
adenocarcinoma cells revealed that though continued>
“many cytoplasmic proteins” were efficiently solubilized in interest to a control, such as a housekeeping protein.
RIPA, some histones and transcription factors were confined But that assumes the housekeeping protein’s abundance
to the insoluble fraction. Cleaved caspase 8, an apoptotic is constant across conditions—an assumption that must be
indicator, was soluble in Laemmli sample buffer (62.5 mM tested. Also, researchers must test to ensure that both the
Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol, housekeeping protein and protein of interest produce linear,
0.01% bromophenol blue), but not in RIPA or NP-40 buffer (50 proportional responses, such that a twofold change in protein
mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, abundance yields a twofold change in signal.
plus inhibitors). Other options include Tris-Triton buffer for In one recent example, Alicia McDonough and colleagues
cytoskeletal proteins (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM at the Keck School of Medicine at the University of Southern
EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, California, loaded a gel with kidney homogenate samples.
0.5% deoxycholate) and 20 mM Tris-HCl pH 7.5 for soluble Some lanes contained 3 mg of the lysate; others contained
cytoplasmic proteins (1). twice as much. The team then probed with antibodies for
claudin-10 and actin. The claudin signal was twice as intense
in the high-abundance lanes, indicating that the protein was in
a linear detection range. But actin, a common control protein,
Researchers can cast their own gels produced an equivalent signal in both cases, indicating that it
“is not an acceptable loading control for this application” (3).
for separating proteins, but they One possible solution is normalizing against multiple
controls. For instance, Janes showed in his study that the
also can buy them precast. That offers
apparent abundance of phosphorylated Smad2 protein
a speed advantage, but it’s also changes dramatically depending on the choice and number
of normalization controls he used (he tested as many as five at
safer and more reproducible. once). “As higher-order combinations of loading controls were
tested as normalizers, I found that the coefficient of variation
In a separate experiment, Janes illustrated the importance of of p-Smad2 linker steadily improved toward 7% to 8%,” he
phosphatase inhibitors during lysis in probing various members wrote (2).
of the “Akt-glycogen synthase kinase-3 (GSK3)-glycogen An alternative approach normalizes each band’s intensity
synthase (GS) signaling axis.” In particular, he showed that to total protein in the lane using either a general protein stain,
some post-translational modifications were more stable in the such as Ponceau S, Memcode, or fluorescent dye. McDonough
absence of inhibitors in NP-40 lysis buffer than RIPA. recommends running a parallel “loading gel, which is loaded
“These results collectively showed that lysis buffer and run identically to the gel(s) that will be immunoblotted” (3).
composition substantially affects the results of quantitative The gel is stained and imaged, and the intensities of several
immunoblotting,” Janes wrote (2). discrete bands averaged for normalization. Alternatively,
the samples can be prelabeled prior to electrophoresis
Normalization and the total signal on the membrane after transfer can
Western blotting typically is used to measure changes be used as loading control. This is especially suitable for
in protein abundance across different conditions. For such fluorescent Western, since the target signals can be detected
comparisons to be meaningful, each lane should contain the simultaneously on the membrane by multiplexing.
same amount of protein, ideally representing equivalent cell
numbers. Electrophoresis and blotting
First, researchers need to know how much protein they have Researchers can cast their own gels for separating proteins,
in each sample. The Abcam guide recommends quantifying but they also can buy them precast. That offers a speed
protein concentration using “a Bradford assay, a Lowry assay advantage, but it’s also safer—unpolymerized polyacrylamide is
or a BCA [bicinchoninic acid] assay,” usually with bovine serum neurotoxic—and more reproducible. Precast gradient gels can
albumin (BSA) as a protein standard (1). be especially useful, providing particularly sharp resolution over
Then, because minor pipetting errors during loading the specific size ranges; such gels are challenging to create from
gel or errors when measuring protein concentration can scratch in a reproducible fashion. Precast gels also are available
dramatically over- or under-emphasize biological effects, for analysis of nondenatured (i.e., “native”) proteins. These are
researchers should also take steps to ensure an equal amount useful especially when studying intact protein complexes or
of protein is present in each lane. using antibodies that recognize conformational epitopes.
In one common approach to minimize the effect of loading Western blots typically use either nitrocellulose or
variation, researchers compare the intensity of their protein of polyvinylidene fluoride (PVDF) membranes for protein blotting.
The latter tends (TBST or PBST) (6),

to be sturdier and but the number,
have a higher duration, and
protein-binding stringency of washes
capacity, but may can vary, as can the
also yield higher amount of detergent
auto fluorescence used. Antibody
depending on dilution also is key—
membrane type. you need sufficient
On the other hand, antibody to detect
nitrocellulose is the target, but not
more compatible so much as to create
with most protein high background on
stains that enable the blot, which can
visual inspection of obscure faint bands.
transfer efficiency. The secondary
Once your antibody is likewise
proteins are critical, so be sure to
transferred to a optimize your dilu-
membrane, blocking tion and incubation
reagents are used to conditions there, as
reduce antibodies well.
nonspecific binding Like all methods,
on the membrane. Common options include nonfat milk and Western blotting has both strengths and weaknesses. The
bovine serum albumin, but several commercial formulations exist technique is easy enough that almost anyone can perform it.
too. In many cases, any blocker will do, but it can be antibody The question is: how well does that signal reflect the cells it
dependent. Milk is suboptimal for phosphorylation analysis, represents? With careful experimental design and rigorous
for instance, because milk contains casein, a phosphoprotein control procedures, researchers can be sure it’s as close as
that can compete with the target for antibody binding and possible.
reduce specific signal intensity. Milk also may contain biotin and
glycoproteins that will light up with streptavidin-based detection Jeffrey M. Perkel is a freelance science writer based in Pocatello,
schemes (4, 5). When in doubt, try several options to see which Idaho.
works best for your system.
References
Antibodies 1. Abcam, “Sample preparation (WB guide),” [http://www.
The key reagent in a Western blot, of course, is the primary abcam.com/index.html?pageconfig=resource&rid=11379
antibody used to recognize the protein of interest. Thousands #A1].
of antibodies are commercially available, but not all are 2. K. A. Janes, Science Signaling 8:rs2 (2015).
equally good. Look for antibodies that have been validated as 3. A. A. McDonough et al., Am. J. Physiol. Cell Physiol. 308 C426
compatible with Western blotting—typically, these recognize (2015).
linear peptides as opposed to conformational epitopes, as 4. LI-COR Biosciences, “Optimizing chemiluminescent
most Westerns are run under denaturing conditions. (Native Western blots,” 2011. [biosupport.licor.com/docs/Chemi_
PHOTO: © PY PHOTO/SHUTTERSTOCK.COM
Westerns, of course, retain protein conformation). You may need Good_Westerns.pdf].

to test several primaries to see which works best, but be sure 5. GE Healthcare Life Sciences, “Western Blotting Principles
also to demonstrate that the antibody actually is specific for and Methods,” 2011. [www.gelifesciences.com/file_source/
your antigen in the first place—that is, that is produces a band of GELS/Service%20and%20Support/Documents%20and%20
the correct size in positive control samples and not in negative Downloads/Handbooks/pdfs/Western%20Blotting.pdf].
controls (3). 6. Cell Signaling Technology, “Western blotting protocol,” [http://
Then, optimize how much and how long blots are incubated www.cellsignal.com/common/content/content.jsp?id=western].
with an antibody and the washing conditions used. Typical
wash conditions are three washes of 5 minutes each in Tris- or
phosphate-buffered saline (TBS or PBS) containing Tween 20
Choosing your
from now, assuming the blot is properly handled and stored.
Chemiluminescent signals, in contrast, are transient and variable.
Researchers performing fluorescence-based Westerns also
detection method have the ability to combine two or more secondary antibodies
to detect multiple proteins at once, a process called multiplex-
ing. Multiplexing—which requires only that the different primary
By Jeffrey M. Perkel antibodies be raised in different organisms and that the corre-
sponding secondaries be conjugated to spectrally distinct dyes—
reduces an experiment’s hands-on time and improves quantita-
tive accuracy and reproducibility relative to chemiluminescence,
which is strictly a singleplex technique.
Quantitative vs. qualitative detection
I
n their seminal Western blotting paper, Towbin et al. devel- By default, Western blotting is inherently qualitative—a protein
oped their blots using radioisotopic labeling, colorimetric is either present, or it is not. But researchers frequently use the
detection, or fluorescence (1). Thirty-six years later, the technique to assess quantitative differences, such as how protein
method has changed remarkably little. But one thing that has abundance changes under different conditions.
changed is how the experiment is read out: Modern researchers Fundamentally, converting a band’s intensity into a reliable
most commonly develop their Western blots using chemilumi- and meaningful assessment of abundance requires that the over-
nescence detection. all amount of the protein does not vary from lane to lane, that the
Fluorescence-based detection—which in Towbin’s study was signal any band generated be linearly related to that protein’s
the least sensitive method tried—has also evolved into an increas- abundance, and that the detector’s ability to capture that signal
ingly sensitive and popular alternative. also is linear. Film, for instance, is a poor choice for quantitative
Both chemiluminescent and fluorescent detection offer sub- Western blotting, writes Kevin Janes of the University of Virginia,
stantial advantages over the previously preferred options of colo- because its dynamic range “is so small that quantitative analysis
rimetry and radiolabeling, being more sensitive than the former is virtually impossible. Film can make small differences in abun-
and less hazardous and troublesome than the latter. But they also dance appear as large differences in band intensity. When satu-
differ from each other in several important ways. rated, film exposures can also hide sample-to-sample variation
for high-abundance proteins such as loading controls” (2; see
Chemiluminescence vs. fluorescence page 12).
In chemiluminescence, the secondary antibody—the antibody One way to ensure band intensity accurately reflects protein
that allows researchers to detect the antibody binding to the pro- abundance, and to account for sample-to-sample variation, is
tein of interest—is conjugated to an enzyme [usually horseradish multiplexing. Rather than assessing protein abundance from
peroxidase (HRP)], just as in Towbin’s original study (1). But where the intensity of a single band in isolation, researchers typically
Towbin and colleagues detected their HRP-tagged antibodies via compare it to one or more invariant normalization controls, which
the deposition of a colored precipitate from a colorless substrate, usually are housekeeping proteins (see Preparing for your West-
chemiluminescence uses hydrogen peroxide to oxidize luminol, ern blot, page 7).
producing a transient burst of photons that must be detected us- In a fluorescent Western, this is easily accomplished. A re-
ing autoradiography or a digital imager. searcher could use a goat anti-rabbit secondary coupled to Cy3
Fluorescence detection uses secondary antibodies labeled to detect their protein of interest, and a rabbit anti-mouse sec-
with fluorescent dyes, which also are detected on a digital im- ondary conjugated to Cy5 (i.e., a dye with distinct excitation and
ager. An alternative approach, chemifluorescence couples HRP emission spectra) to detect, say, tubulin. Using the fluorescence
activity to formation of a fluorescent product. intensity of those two signals, they then can determine the abun-
Chemiluminescent Westerns generally are considered more dance of their protein in their experimental condition relative to
sensitive than their fluorescent counterparts, with detection down the control—a ratio that is more reliably comparable from experi-
to the low femtogram level in some cases. But as a practical mat- ment to experiment than the single protein’s absolute intensity.
ter, fluorescence Western blots are simpler to perform, as there’s The problem is, chemiluminescence doesn’t offer multiple
no chemiluminescent substrate to add (which saves time and detection channels. The only way to quantify multiple proteins in
money) and the signal is generally stable over time (simplifying a chemiluminescent Western is to strip off the primary antibody
data collection and enhancing reproducibility). (using, for instance, 6M guanidine-HCl), and reprobe the blot with
All things being equal, an exposure taken of a fluorescently another primary antibody against the second protein of interest—
labeled blot today will be exactly the same as one taken a week a process that adds time and impacts reproducibility, since
stripping can also remove and colleagues described

target proteins. Alternatively, a quantitative assay for the
researchers can run and probe detection of Plasmodium
replicate blots in parallel, but falciparum circumsporozoite
this, too, is problematic, as the protein (PfCSP), a potential
blots may not be treated iden- vaccine antigen.
tically. The assay, the authors re-
Complicating quantification port, was linear between about
of chemiluminescent Westerns 3 and 12 pg of protein, making
is the detection mechanism it “the most sensitive assay for
itself. Chemiluminescence immunoassay-based detec-
stems from an enzymatic re- tion of PfCSP or any malarial
action that consumes a finite protein.” (ELISA assays, by com-
supply of substrate. Thus, the parison, required at least 100
signal intensity of a chemilu- ng of protein for detection.)
minescent blot will vary with Furthermore, the authors found
time and substrate concentration. A 30 second exposure taken the assay to be highly reproducible, with an inter-assay coefficient
immediately after substrate addition will look very different from of variation (CV) of 10.31% and a mean intra-assay CV of 3.16%
an exposure of the same duration collected 10 minutes later. It (3). But to achieve that level of reproducibility, the authors had to
may also vary if the substrate reagent is a little older, or doesn’t go beyond the typical Western protocol, using serial dilutions of
cover the blot uniformly. As a result, it can be difficult to acquire protein preparations, standard curves, and multiple replicates.
an “ideal” exposure, as it isn’t necessarily obvious how different
exposures will appear. Equipment and reagents
The situation can be particularly tricky in cases involving mul- Other differences between chemiluminescence- and fluo-
tiple bands of dramatically different intensities. At the very least, rescence-based Westerns include the equipment and reagents
researchers may end up wasting considerable time fine-tuning required to run the experiments. Nitrocellulose membranes, for
their exposures to capture both bands in the detector’s linear instance, are compatible with both approaches, but are relatively
range, such that their intensities accurately reflect their abun- fragile. Polyvinylidene fluoride (PVDF) membranes are sturdier,
dance. Experimental timing becomes key in such situations, and but exhibit lower sensitivity and higher autofluorescence. Dedi-
replicating such conditions from day to day requires technical cated “low-fluorescence” membranes are commercially available
skill. Even then, it isn’t easy to compare one “ideal” image with to address these shortcomings.
another captured from a different blot, on a different day, so re- Chemiluminescence can be detected in most standard gel
searchers often are advised against direct comparisons between documentation systems, and even on X-ray film (though that is
experiments. not advised, given film’s poor sensitivity and narrow dynamic
In contrast, fluorescence signals are largely quantitative, so a range). Fluorescence, however, requires a dedicated instrument,
band with twice the intensity of another band can generally be such as a laser scanner or CCD camera equipped with suitable
assumed to contain twice as much material. light sources and emission filters.
In his recent study on the factors influencing the reliability Given all these variables, which option should you choose?
of quantitative Western blotting, Janes wrote that his lab favors In many cases, it really doesn’t matter. Researchers interested in
fluorescence over chemiluminescence Westerns “whenever pos- a qualitative assessment of their samples can often use either
sible” (2). chemiluminescence or fluorescence detection, depending on
PHOTO: © EXTENDER_01/SHUTTERSTOCK.COM
“I compared the linearity of [fluorescent dye]-conjugated the antibodies and equipment they have on hand. But for a deep
secondary antibodies to horseradish peroxidase conjugates that dive into protein quantitation, they may want to give fluorescence
were incubated with an enhanced chemiluminescence (ECL) a try.
cocktail or a commercial substrate marketed for high-sensitivity
applications,” Janes wrote. “Under the same immunoblotting con- Jeffrey M. Perkel is a freelance science writer based in Pocatello,
ditions, chemiluminescent exposures consistently yielded stron- Idaho.
ger band densities. However, the linear dynamic range was very
limited, and signals often decreased at high protein inputs” (2). References
That’s not to say quantitative chemiluminescence is impos- 1. H. Towbin et al., Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979).
sible. In one 2013 report, Sanjai Kumar of the U.S. Food and 2. K. A. Janes, Science Signaling 8, rs2 (2015).
Drug Administration’s Laboratory of Emerging Pathogens, 3. S. Kumar et al., J. Immunol. Methods 390, 99 (2013).
An analysis of critical factors for equal volume of dithiothreitol-containing

Laemmli sample buffer (7), and then immu-
quantitative immunoblotting
noblotted for 20 different protein targets. As
expected, RIPA lysis buffer efficiently solubi-
lized many cytoplasmic proteins [glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH)
Kevin A. Janes and heat shock protein 90 (Hsp90)] and sig-
naling proteins [inhibitor of nuclear factor
Immunoblotting (also known as Western blotting) combined with digital image analysis κB α (IκBα) and various kinases] (Fig. 1, A
can be a reliable method for analyzing the abundance of proteins and protein modifica- and B). RIPA buffer also extracted the cy-
tions, but not every immunoblot-analysis combination produces an accurate result. I toskeletal and cytoskeleton-associated pro-
illustrate how sample preparation, protocol implementation, detection scheme, and teins, actin and focal adhesion kinase (FAK).
normalization approach profoundly affect the quantitative performance of immunoblot- However, tubulin and intermediate filament
ting. This study implemented diagnostic experiments that assess an immunoblot-analysis proteins (lamin A and KRT5) showed sub-
workflow for accuracy and precision. The results showed that ignoring such diagnostics stantial losses into the RIPA-insoluble frac-
can lead to pseudoquantitative immunoblot data that markedly overestimate or underes- tion (Fig. 1C). Remarkably, RIPA insolubility
timate true differences in protein abundance. was not limited to cytoskeletal proteins: the
transcription factor GATA2 and the cell-cell
adhesion protein β-catenin were also pres-
INTRODUCTION difluoride (PVDF) under modified condi- ent in the insoluble fraction. In contrast, ly-
A
mong the most indispensible tools in cell tions of Towbin et al. (4) (25 mM tris, 192 sis with Laemmli sample buffer, followed by
signaling research is the immunoblot. mM glycine, 0.0375% SDS, and 10% methanol shearing of the viscous genomic DNA with a
The premise of immunoblotting is sim- unless otherwise indicated); detection was high-gauge needle, solubilized proteins that
ple, but execution is tricky, and there are performed on either a LI-COR Odyssey instru- are tightly associated with DNA, such as his-
many variations in the method that can ment (for fluorescence detection) or a Bio-Rad tones (Fig. 1D). Despite rules of thumb for
affect the outcome (1). Add quantitation to the ChemiDoc MP gel imager (for chemilumines- protein solubility in various lysis buffers (6),
end of an immunoblot and the complexity of cence detection); and quantitation of raw 16- these results show that it is best to confirm
implementations increases even further. Sur- bit digital images was implemented with the proper solubilization of proteins of interest
prisingly, there are few objective studies on ImageJ gel analysis plug-in (5). Using film to before embarking on an immunoblot study.
quantitative immunoblotting in the primary perform quantitative immunoblotting was If 10 to 30% of a protein were consis-
literature (2, 3). Lacking a systematic assess- avoided entirely, because the dynamic range tently lost in the insoluble fraction, then
ment of key factors, researchers are prone to of film is so small that quantitative analysis is the choice of lysis conditions would not be
repeat or reinforce mistakes that others have virtually impossible (3). Film can make small critical. However, specific proteins can shift
made before them. differences in abundance appear as large dif- between soluble and insoluble fractions in
Here, I analyze how various methodological ferences in band intensity. When saturated, a stimulus-dependent manner. As an ex-
choices affect the ability to perform quantita- film exposures can also hide sample-to-sam- ample, I activated the FAS death receptor
tive immunoblotting accurately and precisely. ple variations in high-abundance proteins in MCF10A-5E human breast epithelial cells
The analysis revealed how seemingly minor such as loading controls. Therefore, through- (8) and lysed the cells in NP-40 buffer (lack-
variations affect immunoblot linearity and out this study, all data were acquired as digi- ing SDS and deoxycholate), RIPA buffer, or
reproducibility, yielding pseudoquantitative tal images. The diagnostic experiments shown Laemmli sample buffer. Although the cleav-
numbers that are not directly proportional here can be easily adapted for other hardware age of caspase-3 was readily detected in all
to the input material. After background sub- and reagent configurations. three preparations (Fig. 2A), cleaved forms
traction, quantitative immunoblots should of caspase-8 were only detected in Laemmli
strive for zero-intercept linearity: y = bx, RESULTS sample buffer (Fig. 2B). Thus, quantitative
where y is the quantified band intensity, x is Sample preparation: A critical factor for measures of caspase-8 processing would re-
the abundance of the protein or modification quire the Laemmli preparation for accurate
quantitative immunoblotting
state in the sample, and b is a proportional- results (9–11). My laboratory has found that
ity coefficient. The value of b is flexible, but The conditions of cell lysis have a profound similar precautions are required for moni-
lines with nonzero intercepts indicate errors impact on the proteins that are extracted and toring regulated changes in intermediate-
in background subtraction, and nonlinear re- the condition in which they are preserved. For filament proteins (12), such as KRT5 (Fig. 1C).
lationships suggest problems with detection example, lysis of cells or tissues with purely Regulated oligomeric or polymeric protein
sensitivity or saturation. Either scenario will nonionic detergents (Triton X-100 or NP-40) assemblies may be particularly susceptible to
yield fold change estimates that are skewed causes some proteins to partition into the differential partitioning between soluble and
relative to the true differences among samples. soluble and insoluble (pellet) fractions after insoluble fractions.
Throughout this work, I systematically centrifugation. Radioimmunoprecipitation The stability and posttranslational modifi-
altered several experimental parameters assay (RIPA) buffer—containing dilute SDS cations of lysate proteins are also affected by
that are often neglected or overlooked when (a denaturing detergent) and deoxycholate (a the activity of co-mingling cellular enzymes,
immunoblotting. Many other parameters disruptor of protein-protein interactions)—is such as proteases and phosphatases. These
were kept fixed: All gels were run as 15- widely used as a lysis buffer for whole-cell ex- enzymes are usually blocked with inhibitors
well, 1.5-mm-thick, tris-glycine minigels on traction. Nonetheless, RIPA buffer lysis still that are supplemented into nondenaturing
a Bio-Rad Protean III platform; all wet elec- generates an insoluble fraction with major lysis buffers, but the SDS and deoxycholate
trophoretic transfers were done onto low- protein constituents from the cytoskeleton in RIPA are sometimes assumed to inacti-
autofluorescence, 0.45-μm polyvinylidene and extracellular matrix (6). vate most cellular enzymes. I tested how ef-
To test how RIPA lysis conditions affected fectively RIPA and NP-40 buffers inhibited
Department of Biomedical Engineering, University of Virginia,
immunoblotting results, I lysed HT-29 hu- protein phosphatases by omitting from both
Charlottesville, VA 22908, USA man colon adenocarcinoma cells in RIPA buffers the Ser-Thr phosphatase inhibitor
E-mail: kjanes@virgnia.edu buffer, boiled the RIPA-insoluble pellet in an microcystin-LR (13) and the Tyr phosphatase
12 Originally published 7 April 2015 in SCIENCE SIGNALING sciencemag.org SCIENCE

SE C T I O N T H R E E | RESEARCH ARTICLES
Fig. 2. Posttranslational modifications can move protein into the

insoluble fraction of common lysis buffers. MCF10A-5E cells were ex-
posed to the Fas cross-linking agent anti-APO-1 (1 μg/ml) (48) for 24 hours,
and then floating and adherent cells were lysed in NP-40 lysis buffer, RIPA
buffer, or dithiothreitol-containing Laemmli sample buffer (SB). (A) Effect
of solubilization conditions on the detection of cleavage (Clv.) products of
caspase-3. (B) Effect of solubilization conditions on the detection of cleav-
age products of caspase-8. Vinculin, tubulin, GAPDH, Hsp90, and p38 were
used as loading controls where indicated. Data are representative of three
Fig. 1. RIPA buffer solubilizes many, but not all, cellular proteins. (A) experiments.
Examples of proteins that are entirely solubilized [100% in the superna-
tant (Sup)]. (B) Examples of proteins that are mostly solubilized (>90%
methanol percentage conjugated secondary antibodies to horserad-
Sup). (C) Examples of proteins that are partially solubilized (≤90% Sup).
affects quantitative ish peroxidase conjugates that were incubated
(D) Dimethyl-lysine 4 histone H3 (H3K4me2) resides almost entirely in the
accuracy and dynamic with an enhanced chemiluminescence (ECL)
RIPA-insoluble pellet (Pel). Band intensities were quantified from the 16-bit
range of immunoblot cocktail (16) or a commercial substrate mar-
digital image by densitometry in ImageJ and are shown normalized to the
band intensities is keted for high-sensitivity applications (file
Sup lane for each target. n.d., not detected. Data are representative of two
unknown. S2). Under the same immunoblotting condi-
to four experiments.
Using HT-29 cell tions, chemiluminescent exposures consis-
extracts, I performed tently yielded stronger band densities (Fig.
inhibitor orthovanadate (14). I lysed parallel an extended twofold serial dilution from a 5, A to C). However, the linear dynamic range
cultures of AC16 ventricular cardiomyocytes grossly overloaded sample (200 μg of extract) was very limited, and signals often decreased
(15) and immunoblotted for multiple phos- to one below the limit of detection (100 ng of at high protein inputs (Fig. 5C). This can oc-
phorylation sites along the Akt–glycogen extract). Two replicate gels were transferred cur when side products of the peroxidase-cat-
synthase kinase-3 (GSK3)–glycogen synthase in buffer containing 10 or 20% methanol, and alyzed reaction are oxidized and precipitated,
(GS) signaling axis (Fig. 3, A to D). For these then membranes were probed for seven differ- causing the membrane to “brown out” and
phosphoproteins, the addition of phospha- ent targets (file S1). For actin and p38, I found absorb the emitted photons. Titrating down
tase inhibitors was more critical in RIPA buf- that detection was linear up to 50 μg of total the amount of protein or primary antibody
fer than in the nondenaturing NP-40 buffer. protein, irrespective of the methanol concen- can avoid the problem, provided that the re-
The extent of sensitivity depended strongly tration (Fig. 4, A and B). This zero-intercept searcher is aware of it.
on the phosphorylation site, with Thr308 linearity is ideal, because band density is di-
Optimizing loading controls
of Akt, Ser21 of GSK3α, and Ser9 of GSK3β rectly proportional to input material without
showing greater lability than Ser473 of Akt the need to refer to a calibration curve. By Arguably, the biggest source of confusion in
and Ser641 of GS. These results collectively contrast, Hsp90 and tubulin showed a hy- quantitative immunoblotting is the role of
showed that lysis buffer composition sub- perbolic saturation under both transfer con- protein loading and loading controls (17). Nor-
stantially affects the results of quantitative ditions with less than 25 μg of total protein malizing for cell numbers across samples is
immunoblotting. (Fig. 4, C and D). Saturation can result from challenging, because it is difficult to estimate
steric crowding of antibody epitopes, quench- changes in cell proliferation and death under
Assessing immunoblot protocols by
ing of fluorescently labeled secondary anti- different conditions. Such estimates also do
serial dilution
bodies, or oxidation of enzyme-conjugated not account for variations in initial seeding
For immunoblotting, a single protocol that is secondary antibodies. Regardless of the density and final lysate volumes, which will
optimal for all electrophoresis transfer setups and source, band densities in this regime no lon- affect the observed protein abundance. Con-
detection methods does not exist. However, the ger provide a linear estimate of sample abun- sequently, immunoblot samples are often pre-
quantitative accuracy and dynamic range of any dance, and calibration is required to obtain pared according to total cellular protein (18,
protocol can be assessed using a serial dilution accurate measurements. The improved trans- 19), assuming that the average protein con-
of cell extract and a panel of primary anti- fer of proteins in 20% methanol appeared to tent per cell is constant across the different
bodies. As an example, I sought to determine shift the detection of multiple targets from a conditions.
wet-transfer conditions (specifically, metha- linear regime to one of hyperbolic saturation To complement total protein estimates, im-
nol concentration) that enabled quantitative (Fig. 4, E to G). The results from this diagnos- munoblots typically include loading control
detection of most target proteins. Here, the tic study indicated that the lower methanol proteins, which provide a secondary check
transfer buffer always included 10% methanol concentration was preferred for my immu- that roughly equal amounts of cellular mate-
(see Materials and Methods), but other proto- noblot protocol. rial have been added. Two key assumptions of
cols use 20% methanol according to the origi- Similar comparisons have also caused my the loading control are that (i) its abundance
nal conditions of Towbin et al. (4). Although laboratory to favor fluorescence-based de- is roughly constant across the different condi-
immunoblot bands are brighter and crisper tection over chemiluminescence whenever tions, and (ii) its immunoblot band intensity
with the higher methanol concentration, how possible. I compared the linearity of IRDye- is linearly reflective of its abundance. How-
ever, a single loading control may not fulfill Fig. 3. Phospha-

both of these assumptions (2). tase inhibitors are
If total cellular protein can be quanti- critical to preserve
fied accurately, then total protein is loaded certain phos-
equivalently across samples, and one or two phorylated residues
loading controls suffice as a qualitative confir- under certain lysis
mation of overall protein abundance (20–22). conditions. (A and
Conversely, if the total cellular protein is not B) Effect of lysis buf-
known or cannot be determined accurately, fer and presence or
then the input must be normalized to some absence of phospha-
estimate of protein loading. A common ap- tase inhibitors (PPIs)
proach found in the literature is to normalize on the detection of
by only one loading control, but this scaling phosphorylated Akt
is highly problematic. Taking one unknown (p-Akt) on Thr308
quantity (the protein of interest) and divid- (T308) and Ser473
ing it by another unknown quantity (a single (S473). (C) Effect of
loading control) creates a number with very lysis buffer and pres-
poor statistical properties, including an un- ence or absence of
defined mean. The dangers of single-variable PPIs on detection of
normalization have long been recognized in GSK3α/β phosphory-
data from microarrays (23) and quantitative lated on Ser21 and
polymerase chain reaction (PCR) (24), but not Ser9 (p-GSK3α/β).
in data from immunoblotting. A solution is to (D) Effect of lysis
aggregate the band intensities from multiple buffer and presence
loading controls, calculating a mean estimate or absence of PPIs
of total cellular content that is less sensitive on detection of GS
to the technical or biological fluctuations of a phosphorylated on Ser641 (p-GS). AC16 cells were lysed in RIPA or NP-40 lysis buffer with or without
single loading control (12, 25). PPIs. Vinculin, tubulin, GAPDH, and actin were used as loading controls where indicated. Total Akt,
To demonstrate the utility of multiprotein GSK3α/β, and GS were used to monitor specific changes in protein abundance. Band intensities were
normalization, I immunoblotted for linker quantified from the 16-bit digital image by densitometry in ImageJ and are shown normalized to the
phosphorylation of Smad2 on Ser245/250/255 (p- average +PPI conditions for each target across both lysis conditions. Data are representative of two
Smad2 linker) in MCF10A-5E cells that had experiments.
been stimulated with transforming growth
factor–β (TGFβ) (file S3). Under these condi- ficient of variation by more than twofold (21 normalization because of its reduced sample-
tions, Smad2 linker phosphorylation is medi- to 9%), but normalizing to tubulin had virtu- to-sample variation. An alternative strategy
ated by cyclin-dependent kinases (CDKs) (26), ally no effect (Fig. 6C). This does not imply is to prepare a separate set of immunoblot
so cells were additionally pretreated with or that GAPDH is always a good loading control lanes with decreased protein content for
without the pan-CDK inhibitor flavopiridol or that tubulin is always a bad one; rather, it loading controls (2). However, normalizing
(27). The TGFβ stimulation ± flavopiridol emphasizes the danger of relying on a single from different lanes will miss lane-specific
inhibition experiment was performed in bio- measured variable to estimate total protein irregularities in sample preparation or elec-
logical quadruplicate to assess reproducibility content. As higher-order combinations of trophoretic transfer, which can be minimized
of p-Smad2 linker quantification. Lacking any loading controls were tested as normalizers, with good technique but not eliminated.
total protein normalization, p-Smad2 linker I found that the coefficient of variation of p- A possible alternative to multiple load-
densitometry was variable, with flavopiridol Smad2 linker steadily improved toward 7 to ing controls is to use reversible total protein
producing only a marginally significant de- 8%, consistent with values reported previ- stains that are compatible with PVDF mem-
crease in phosphorylation (Fig. 6, A and B). ously (10, 11). This reproducibility became branes (such as MemCode or Ponceau S).
To improve reproducibility, I blotted the same less dependent on the specific combination For a PVDF-compatible stain to be useful, it
membrane for five potential loading controls: of loading controls (note the shrinking error must be quantitative for total cellular protein
total Smad2, tubulin, Hsp90, GAPDH, and bars in Fig. 6C), indicating that I had con- (directly proportional or, at least, hyperboli-
p38. The antibodies for these proteins are verged on a measure of cellular content per cally saturating), and it must not interfere
from various hosts and yield single immuno- lane that was truly representative. With all with the subsequent immunoblot. I tested
reactive bands under the blotting conditions five loading controls, both the TGFβ-induced two total protein stains that can be revers-
used here. The properties of the antibodies stimulation of p-Smad2 linker and its inhibi- ibly applied to PVDF membranes: MemCode
and the detected control proteins enabled tion by flavopiridol were highly significant Reversible Protein Stain (commercially avail-
multiplex detection of loading controls to- (Fig. 6D). able from Thermo Scientific) and Ponceau S
gether with p-Smad2 linker or after a single Others have noted that many common (28). MemCode yielded a strong blue banding
round of membrane stripping (see Materials loading controls are abundant proteins (for pattern that was readily detected by white-
and Methods). example, tubulin) that result in saturated light epi-illumination and a charge-coupled
Upon quantifying band densities in band intensities under the conditions needed device (CCD) camera (fig. S1A). Using a blank
ImageJ, I compared the reproducibility to detect proteins or modification states of in- region of the PVDF membrane to define a
of p-Smad2 linker after normalization to all terest (2). On the basis of the results with the background for subtraction, I found that to-
possible combinations of loading controls: 25 serial dilutions (Fig. 4D), saturation is prob- tal MemCode band intensity increased hyper-
= 32 combinations. For single loading con- ably the reason why tubulin worked poorly as bolically with zero intercept over a relevant
trol normalization, the effect on replicate-to- a loading control for p-Smad2 linker in this range of lysate amounts (fig. S1B). However,
replicate reproducibility heavily depended on setting (Fig. 6B). However, in the context of the staining procedure markedly increased
the choice of loading control. Normalizing to multiple loading controls that are averaged, the 700-channel background fluorescence of
GAPDH decreased the p-Smad2 linker coef- a saturated loading control negligibly affects the membrane, and this background was not
Fig. 4. Linearity and hyperbolic saturation

of immunoblots determined by serial dilu-
tion. (A and B) Immunoblots for actin and p38
are linear under both transfer conditions. (C
and D) Immunoblots for Hsp90 and tubulin are
hyperbolically saturated under both transfer
conditions. (E to G) Linear detection of immu-
noblots for E-cadherin, ERK1/2 (extracellular
signal–regulated kinases 1 and 2), and GAPDH
occurred with tank transfer conditions contain-
ing 10% methanol. HT-29 cells were lysed in RIPA
buffer, immunoblotted for the indicated targets,
and imaged. Left: Immunoblots. Middle: Log-log
plots of the quantified band intensities from the
blots on the left. Right: Linear plots of the same
data. Linear fits are gray when the hyperbolic
model is no better than the linear model for that
transfer condition. Linear fits are red when the
linear fit of the associated transfer condition is
better than the linear fit of the other transfer
condition. Hyperbolic fits are green when the
hyperbolic model is better than the linear model
for that transfer condition. Data are in blue when
neither the linear nor the hyperbolic model pro-
vides a better fit. Model comparisons were done
by the F test [false discovery rate (FDR) = 5%;
n = 5 to 8 dilutions]. See file S1 for raw images
and calculations.
removed by the recommended stain removal

(erasing) procedure (fig. S1C). Although po-
tentially useful for immunoblots detected by
chemiluminescence, MemCode is not suitable
for two-color fluorescence detection.
I uncovered a different set of problems with
Ponceau S. Compared to MemCode, the red
Ponceau S stain was not as efficiently detected
by the CCD camera (fig. S1, A and D). How-
ever, its image densitometry was linear as a
function of lysate amount, and there was no
background fluorescence caused by Ponceau
S staining or erasure (fig. S1, E to G). Pon-
ceau S appeared to fill all the requirements
for a total protein stain, except for one major
drawback—the zero intercept of its densitom-
etry could not be accurately estimated from a
blank region of the PVDF membrane (fig. S1D,
lane B), causing a negative bias of ~10,000 in-
tensity units (fig. S1E). Without a lysate cali-
bration curve to estimate this bias on a PVDF
membrane, I concluded that Ponceau S can-
not be used for relative protein quantification.
When immunoblotting for phosphoproteins,
there are additional complications with eras-
ing Ponceau S from PVDF membranes, be-
cause the alkaline conditions for erasure will
chemically modify phosphorylated Ser and
Thr residues (29).
Given these data with the reversible total
protein stains, accurate quantitation of im-
munoblot data should adopt the best prac-
tices of quantitative PCR (24) and use an
assortment of three or more loading controls,
spanning a range of abundances, when di-
rect total protein measures are lacking. To
minimize cost and effort, my laboratory de-
tects constitutively produced proteins with
high-affinity antibodies that work reliably

at low concentrations (25 to 100 ng/ml),
yield single immunoreactive bands, and
thus are ideal for multiplexing (see Mate-
rials and Methods; Table 1).
Stripping, reprobing, and the total
protein control
Aside from loading controls for total cel-
lular content, immunoblots that quan-
tify protein modification states should
contain an additional control: an immu- Fig. 5. Quantitative immunoblotting is challenging when imaging by chemiluminescence. (A to C)
noblot for the total protein. This control HT-29 lysates were prepared as in Fig. 4, immunoblotted for the indicated proteins, and imaged by IRDye
serves to gauge how much of the observed fluorescence, ECL, or SuperSignal West Femto chemiluminescence as described (12, 16, 45, 46). Linear
change in protein modification can be fits are shown in gray when the hyperbolic model is not significantly better than the linear model for that
explained by differences in target abun- imaging condition. Linear fits are shown in red when the linear fit of the associated imaging condition is
dance. For rapid experiments that are significantly better than that of the other imaging conditions. Hyperbolic fits are shown in green when the
expected to avoid protein turnover and hyperbolic model is significantly better than the linear model for that imaging condition. Data are interpo-
synthesis (such as the one shown in Fig. lated in blue when neither the linear nor the hyperbolic model provides a better fit. All model comparisons
6), the total protein control can addition- were done by the F test at an FDR of 5% (n = 4 to 8 dilutions). See file S2 for raw images and calculations.
ally contribute to a panel of loading con-
trols if changes in band intensity are clearly sidual GAPDH antibody staining. By contrast, Recombinant ERK2 and p38 proteins were
cofluctuating with other loading controls. the guanidinium strip removed most of the cloned, purified, and quantified by protein
However, for comparisons on long time scales, GAPDH antibody but left a clear p-ERK1/2 assay. Using a known mass of bovine serum
across different cell types, or with rapid pro- artifact in the total ERK1/2 reprobe, with in- albumin (obtained as a calibrated standard
tein turnover, the total protein control is spe- creased staining in the +EGF lane. The SDS from a protein assay kit), a standard curve
cifically important on its own and may not be plus β-mercaptoethanol strip completely re- was constructed by running serially diluted
a reliable indicator of loading. moved the GAPDH antibody and yielded the bovine serum albumin on a polyacrylamide
A common way to estimate target protein closest approximation of total ERK1/2 abun- gel alongside recombinant glutathione S-
abundance from a modification-specific im- dance, although with substantial loss of total transferase (GST)–tagged purified ERK2 and
munoblot is to strip the membrane of anti- protein from the membrane based on the im- p38 (22) (Fig. 8A). The gel was then stained
body and then reprobe with an antibody that munoreactivity of the three additional load- with Coomassie blue and scanned for its near-
recognizes the total protein (or at least the un- ing controls (compare the glycine-stripped infrared fluorescence (34), yielding a digital
modified form). Essential to this approach is membrane to the membrane stripped with image for densitometry in ImageJ (see Ma-
confirming that the stripping conditions have SDS plus β-mercaptoethanol). Moreover, after terials and Methods). To accommodate some
fully removed the original modification-spe- SDS plus β-mercaptoethanol stripping, the to- degree of band saturation and improve the
cific antibody from the membrane of choice tal ERK1/2 bands still showed an artifactual dynamic range of detection, I fit the protein
(PVDF or nitrocellulose). If not, the reprobed increase in the EGF-treated sample. To avoid band intensities to a simple hyperbolic curve:
blot will show artifacts from the residual first the need to strip and reprobe the same blot,
antibody. As an example, I acutely stimulated an alternative is to use two-color fluorescence a × Protein
AC16 cells with epidermal growth factor (EGF) detection with phosphorylation-specific and Intensity =
b + Protein
for 5 min and immunoblotted for phosphory- total antibodies that are raised in different
lated ERK1/2 (p-ERK1/2) with an antibody hosts and against different epitopes (Fig. 7C). with two free parameters (a and b) that were
that is difficult to remove. Technical replicates Another option is to measure total target pro- estimated by least-squares regression. The
of the same two lysates showed good repro- tein by immunoblotting a replicate set of sam- modeled fit captured all of the albumin stan-
ducibility in the observed EGF-stimulated ples and confirming that the matched loading dards and enabled mapping the measured
induction of p-ERK1/2, and loading controls controls are comparable to those in the modi- GST-ERK2 and GST-p38 band densities to
were consistent across the membrane (Fig. fication-specific immunoblot (Fig. 7D). total protein amount (Fig. 8B). Dividing
7A). After cutting the membrane into thirds, I by the volume of sample loaded into the
Advanced quantitative immunoblotting:
tested three stripping conditions: (i) a gentle gel resulted in an estimated concentration
Absolute quantification
low-pH glycine buffer solution [1.5% glycine of full-length protein in the recombinant
(pH 2.2), 0.1% SDS, and 1% Tween], (ii) a more For some computational models of biochemi- preparation.
stringent guanidinium solution (6 M guani- cal networks, the absolute abundances of Size separation of the recombinant protein
dine-HCl) (30), and (iii) a high-stringency SDS specific cellular proteins are needed (32, 33). before quantification is critically important,
plus β-mercaptoethanol solution with heat Such applications require explicit calibra- because purified preparations often contain
[62.5 mM tris (pH 6.8), 2% SDS, and 100 mM tion using proper absolute standards on the cleavage products that add to total protein
β-mercaptoethanol at 50°C] (31). The stripped same immunoblot. For the cellular proteins but are not immunoreactive. Commercial
membranes were blocked identically and rep- of interest, appropriate standards are puri- vendors of recombinant protein may simply
robed for total ERK1/2 along with three addi- fied recombinant proteins, which have been quantify total protein (full length plus frag-
tional loading controls. Reprobed membranes calibrated against a purified protein of known ments that are not useful for calibration),
were imaged and displayed identically to em- mass. ultimately resulting in an overestimation of
phasize differences in the immunoblot signal To illustrate this process, I quantified the cellular protein. For calibration, one should
detected (Fig. 7B). absolute masses of ERK2 and p38 (per 25 μg also remember that unknown samples must
The experiment revealed that the low-pH of cellular extract) in HT-29 and AC16 cells fall between calibration samples that are well
glycine strip was ineffective at removing the (file S4). Absolute quantification enabled fit by the linear or hyperbolic curve. Samples
antibodies. Indeed, “total” ERK1/2 looked ERK2-p38 comparisons within each cell line that fall outside the calibration range will
identical to p-ERK1/2, and there was even re- as well as between cell lines. cause the linear or hyperbolic equation to
make extrapolations that could persistent concern is saturation

be highly inaccurate. (Fig. 4). Saturated immunoblots
Before making comparisons do not overestimate a change
between cell lines, I verified in protein; instead, they can
that loading the same total mass substantially underestimate it.
of HT-29 and AC16 extract gave To demonstrate, I plotted dilu-
approximately equivalent band tions of an unstimulated and
intensities for several load- stimulated extract for a theo-
ing controls (Fig. 8C). Hsp90 retical immunoblot band that
abundance was higher (per is hyperbolically saturated (Fig.
total mass) in HT-29 cells, 9). There is a clear threefold
whereas vinculin and tubulin change in abundance when the
were higher in AC16 cells, and target is immunoblotted under
GAPDH and actin were approxi- conditions where the linear ap-
mately equal. The uncorrelated proximation is accurate (<10
abundance of these “housekeep- μg of extract in this example).
ing proteins” suggests that the However, overloading the gel
cell extracts can be fairly com- with 50 μg of extract reduces
pared on a total protein basis. the difference to 1.4-fold. This
The results further reinforced type of dampening has led some
the importance of using mul- to conclude that fluorescence
tiple loading controls. detection is not sensitive com-
To quantify the protein of pared to chemiluminescence
interest in cellular extracts, I (especially on film). However,
ran the calibrated standards the comparison is not fair if the
and the extracts alongside one 1.4-fold difference in antibody
another and blotted with an binding has been exaggerated
antibody that detects both the by a nonlinear chemilumines-
recombinant protein and the Fig. 6. Reproducibility of quantitative immunoblots across biologi- cent reaction (Fig. 5, B and C).
protein of interest (Fig. 8, D to cal replicates is improved after normalization to multiple loading The analysis here also il-
G). Note that the ERK2 calibra- controls. (A) Representative immunoblot for phosphorylated Smad2 on lustrated that quantifying
tion cannot be used to quantify Ser245/250/255 (p-Smad2 linker) in MCF10A-5E cells stimulated with TGFβ (50 phosphorylation-specific immu-
the ERK1 band at 44 kD because ng/ml) for 30 min with or without 1 hour of preincubation with 300 nM fla- noblots as a “phosphorylated-to-
of differences in the immuno- vopiridol. Tubulin, Hsp90, GAPDH, and p38 were used as loading controls. total ratio” is fraught with both
reactive epitope. In addition, Total Smad2 was used to monitor overall changes in protein abundance numerical complications (Fig. 6)
a mass correction is needed and served as a fifth candidate loading control for this analysis. (B) Raw and potential experimental arti-
to account for the size differ- p-Smad2 linker densitometry quantified in ImageJ. (C) Decrease in the co- facts (Fig. 7). The ratio is further
ence between the GST-tagged efficient of variation among p-Smad2 biological replicates with increasing prone to be misinterpreted as a
recombinant protein and the numbers of loading controls. The best (GAPDH) and worst (tubulin) single phosphorylation stoichiometry,
endogenous protein, because loading control normalizations are highlighted. (D) p-Smad2 linker densi- which cannot be calculated in
immunoblotting quantifies an- tometry after normalization to the mean band intensity of tubulin, Hsp90, experiments that use differ-
tigen independently of its mass GAPDH, p38, and total Smad2 for each biological replicate. For (B) and (D), ent antibodies to detect the
(the correction is 42/69 for data are shown as means ± SE of n = 4 biological replicates, with differences phosphorylated and total pro-
ERK2 and 38/65 for p38; Fig. 8, in means assessed by Welch’s two-sided t test. For (C), data are shown as tein because of differences in
E and G). This analysis showed mean coefficients of variation ± SD of n = 1 to 10 possible normalization antibody affinity. Calculating
that AC16 cells had greater than combinations for the indicated number of loading controls. See file S3 for phosphorylation stoichiometry
sevenfold more ERK2 protein raw images and calculations. by immunoblotting requires
compared to HT-29 cells, despite electrophoretic conditions that
only about twofold difference in separate the phosphorylated
band intensity (Fig. 8E). The limited differ- to be meaningful (8, 36–40). What are the and total forms by mobility (41). Modifica-
ence in band intensity was due to the satura- implications of pseudoquantitative immu- tion stoichiometry can be critical under
tion of the ERK2 immunoblot at high protein noblotting? When chemiluminescence is certain conditions (42), but there are also
concentrations, which was captured by the used cavalierly, there is a danger of wildly examples where increases in total protein
calibration curve. ERK2 abundance in AC16 exaggerated claims. For example, a twofold have affected signal flow (43). Considering
cells was also high compared to the abun- change in Hsp90 abundance might appear these caveats, my conclusion is that phos-
dance of p38, which had a concentration that as a fivefold change in the intensity of the phorylated-to-total ratios based on stripped
was an order of magnitude lower (Fig. 8G). band on an immunoblot (compare the ECL and reprobed membranes should be avoided
Although laborious, this type of absolute values for ~6 and ~3 μg in Fig. 5B). A two- with immunoblot data.
quantification can be done systematically for fold change in GAPDH abundance could be A major challenge in evaluating the im-
multiple cellular proteins and provide new “quantified” as a >20-fold change on the munoblot data of others is that publications
insight into signaling function (35). basis of band intensity (compare the ECL and manuscripts will often omit details on
values for 25 and ~13 μg in Fig. 5C). This acquisition that are considered “routine.”
DISCUSSION makes numerical results look very impres- Those details matter, because the evalu-
Just because we can put numbers on an im- sive, but the truth is still a twofold change ation criteria of an immunoblot detected
age does not imply that we should—a quan- in abundance. by film are different than those of an im-
tified biomolecule should relate directly to With fluorescence detection, there is not munoblot detected by fluorescence. When
the true quantity of that biomolecule if it is the danger of a runaway reaction, but a such information is missing, readers and
Fig. 7. Membrane stripping and reprobing is a quantitative trade-off between antibody removal and total protein loss. (A) Replicate immunoblots
for ERK1/2 phosphorylated on Thr202 and Tyr204 of ERK1 and Thr185 and Tyr187 of ERK2 (p-ERK1/2) in AC16 cells stimulated with EGF (100 ng/ml) for 5 min.
GAPDH and tubulin were used as loading controls in the first immunoblot. (B) Reprobe of the membrane in (A) for total ERK1/2 after stripping with glycine
buffer, guanidinium, or β-mercaptoethanol (βME) stripping buffer. Vinculin, Hsp90, and actin were used as loading controls for the reprobed blots. (C)
Two-color fluorescence immunoblot for p-ERK1/2 (green) and total ERK1/2 (magenta) of the same lysates as in (A). Vinculin and Hsp90 were used as
loading controls. (D) Direct immunoblot for total ERK1/2 of the same lysates as in (A). A lower percentage polyacrylamide gel was used in (C) and (D) to
emphasize the total ERK1/2 upshift after stimulation with EGF. GAPDH, vinculin, Hsp90, and tubulin were used as loading controls. Data are representative
of two experiments.
Fig. 8. Workflow for absolute protein quantification. (A) Serial dilution of an albumin standard to calibrate recombinant purifications of GST-ERK2
and GST-p38 by Coomassie staining. (B) Albumin band intensity (black) plotted as a function of protein and fit to a hyperbolic model (gray) that infers
the amounts of GST-ERK2 (green) and GST-p38 (purple) proteins. (C) HT-29 and AC16 cells have roughly equal protein constituents by mass based on
the amount of Hsp90, vinculin, tubulin, GAPDH, and actin detected in 25 μg of each sample. (D) Serial dilution of the GST-ERK2 standard to calibrate en-
dogenous abundances of ERK2 in HT-29 and AC16 cells. (E) GST-ERK2 band intensity (black) plotted as a function of protein input and fit to a hyperbolic
model (gray) that infers the amount of ERK2 in HT-29 cells (blue) and AC16 cells (red). (F) Serial dilution of the GST-p38 standard to calibrate endogenous
abundances of p38 in HT-29 and AC16 cells. (G) GST-p38 band intensity (black) plotted as a function of protein input and fit to a hyperbolic model (gray).
The model was used to infer the amount of ERK2 in HT-29 cells (blue) and AC16 cells (red). Data are representative of two experiments. See file S4 for raw
images and calculations.
reviewers can distinguish a film exposure ways inappropriate. Rather, I want to convey was isolated and maintained as previously
by the hazy gray of the background and the that with numbers comes great responsibil- described (8). AC16 cells (15) were purchased
blurred borders of bands resulting from the ity. There are straightforward ways to diag- from M. Davidson (Columbia University) and
flatbed optical scan of a film at arbitrarily nose immunoblot accuracy (Figs. 4, 5, and maintained in Dulbecco’s modified Eagle’s
high resolution (44). Digitally acquired im- 8) and precision (Fig. 6). We should all be medium/F-12 medium (Life Technologies)
munoblots will often have a whiter back- encouraged to complete these diagnostics plus 12.5% tetracycline-free fetal bovine se-
ground with crisper bands that may appear on our own targets and immunoblot setups rum (Clontech) and penicillin-streptomycin
pixelated because of binning on a CCD cam- before diving in to generate “real data.” The (Gibco).
era or the step size of a fluorescence scanner. stakes are simply too high to do otherwise. Cells were stimulated with the indicated
Although some may find them less aestheti- concentrations of anti-APO-1-3 cross-linking
cally appealing, digitally acquired images antibody (Axxora), TGFβ (PeproTech), or EGF
provide the more accurate representation of MATERIALS AND METHODS (PeproTech) for the indicated times, washed
band intensity and its relationship to sample with ice-cold PBS (phosphate-buffered saline),
Cell lines, stimulation, and lysis
abundance. and then lysed in RIPA buffer [50 mM tris-HCl
The message of this research resource HT-29 cells were obtained from the American (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5%
is not that chemiluminescence cannot be Type Culture Collection and maintained as sodium deoxycholate, 0.1% SDS, 5 mM EDTA
quantitative or that film exposures are al- recommended. The 5E clone of MCF10A cells supplemented with aprotinin (10 μg/ml), leu-
peptin (10 μg/ml), pepstatin (1 μg/ml), 1 mM immunoblots here, some phosphorylation

phenylmethylsulfonyl fluoride (PMSF), micro- site–specific antibodies can be competed
cystin-LR (1 μg/ml), and 200 μM sodium or- away from their target epitopes with PBS
thovanadate], NP-40 buffer [50 mM tris-HCl buffers. In this circumstance, the 0.5× block-
(pH 8.0), 150 mM NaCl, 0.5% NP-40 substitute, ing buffer should be prepared with Odyssey
5 mM EDTA supplemented with aprotinin (10 blocking buffer (LI-COR; # 927-50000) and
μg/ml), leupeptin (10 μg/ml), pepstatin (1 μg/ diluted in an equal volume of tris-buffered
ml), 1 mM PMSF, microcystin-LR (1 μg/ml), saline (TBS) throughout the procedure. TBS
and 200 μM sodium orthovanadate], or di- buffers were also used for the fluorescence-
thiothreitol-containing Laemmli sample buf- chemiluminescence comparison (Fig. 5).
fer [62.5 mM tris-HCl (pH 6.8), 2% SDS, 10% All blocking steps used a surface-to-volume
glycerol, 100 mM dithiothreitol, and 0.01% ratio of 5-ml 0.5× blocking solution per 2
bromophenol blue]. Laemmli sample buffer ¾-inch × 3 ¼-inch membrane from a 15-well
lyses were performed at room temperature minigel. The membrane and 0.5× blocking
to avoid precipitating the SDS, and viscos- solution were sealed in a plastic bag and in- Fig. 9. Quantifying partially saturated im-
ity of these lysates was reduced by passing cubated on a rotating platform for 1 hour at munoblots can markedly underestimate dif-
them vigorously through a 25-gauge needle 5 room temperature. ferences between samples. In this theoretical
to 10 times. For RIPA and NP-40 lysates, pro- example, a serial dilution is performed with un-
Immunoblotting: Antibody probing stimulated and stimulated extracts. The relative
tein concentrations were determined with
the bicinchoninic acid assay (Thermo Fisher After blocking, membranes were incubated change in the linear range (b) of the immunoblot
Scientific). with 0.5× blocking solution + 0.1% Tween is [99 (blue)/33 (red)] ~ threefold, whereas the
For solubilization comparisons (Fig. 1), 20 containing primary antibodies recogniz- relative change at fivefold higher loading is only
HT-29 cells were lysed in RIPA buffer supple- ing the proteins or epitopes listed in Table 1. 1.4-fold (36%).
mented with protease and phosphatase in- All primary antibody steps used a surface-
hibitors as described above. After incubation to-volume ratio of 5-ml primary antibody tion per 2 ¾-inch × 3 ¼-inch membrane from
on ice and centrifugation, the RIPA-insoluble solution per 2 ¾-inch × 3 ¼-inch membrane a 15-well minigel. The membrane and second-
pellet was boiled for 5 min in an equivalent from a 15-well minigel. The membrane and ary antibody solution were sealed in a plastic
volume of dithiothreitol-containing Laemmli primary antibody solution were sealed in a bag and incubated on a rotating platform for
sample buffer. plastic bag and incubated on a rotating plat- 1 hour at room temperature.
form overnight at 4°C. Membranes were removed from secondary
Immunoblotting: Polyacrylamide gel
Antibody pairs raised in different species antibody solution and washed on a rocking
electrophoresis
were routinely multiplexed when using two- platform for 4 × 5 min in ~25 ml of PBS-T.
Immunoblotting was performed as described color fluorescence detection. In addition, To remove residual Tween 20, which is highly
(12, 45, 46), but the details of the implemen- primary antibodies with negligible off-tar- autofluorescent upon 700-nm excitation, the
tation will be elaborated upon here. Samples get bands could be multiplexed in the same membrane was washed for 5 min in ~25 ml
were prepared in dithiothreitol-containing detection channel if the molecular weights of PBS before scanning. Fluorescence images
Laemmli sample buffer to a total volume of of their protein targets could be clearly re- were obtained on an Odyssey infrared scan-
20 or 40 μl. Polyacrylamide gels (8, 10, 12, or solved from one another. This single-color ner (LI-COR) at 169-μm resolution and 0-mm
15%) of 1.5 mm thickness were cast according multiplexing enabled concurrent detection focus offset. Fluorescence channel intensi-
to (1), and samples were electrophoresed in of multiple loading controls. Common pri- ties ranged from 5.0 to 8.5 depending on the
tris-glycine running buffer (25 mM tris base, mary single-channel combinations included immunoblot.
250 mM glycine, and 0.1% SDS) at 130 V until antibodies recognizing tubulin (50 kD) +
Immunoblotting: Chemiluminescence
the dye front reached the end of the gel. GAPDH (36 kD), Hsp90 (90 kD) + p38 (38
detection
kD), vinculin (120 kD) + actin (42 kD), and
Immunoblotting: Electrophoretic
vinculin (120 kD) + tubulin (50 kD) + GAPDH Membranes were removed from primary an-
transfer
(36 kD). tibody solution and washed on a rocking plat-
Proteins from the polyacrylamide gel were form for 4 × 5 min in ~25 ml of TBS + 0.1%
Immunoblotting: Fluorescence
transferred to a PVDF membrane (Milli- Tween 20 (TBS-T). After washing, membranes
detection
pore; Immobilon-FL, 0.45-μm thickness) in were incubated with 0.5× blocking solution
a Mini Trans-Blot Electrophoretic Transfer Membranes were removed from primary anti- + 0.1% Tween 20 containing horseradish per-
Cell (Bio-Rad) under modified conditions of body solution and washed on a rocking plat- oxidase–conjugated goat anti-rabbit (Jackson
Towbin et al. (4) (25 mM tris, 192 mM gly- form for 4 × 5 min in ~25 ml of PBS + 0.1% ImmunoResearch; #111-035-144, 1:10,000)
cine, 0.0375% SDS, and 10% methanol unless Tween 20 (PBS-T). After washing, membranes or anti-mouse (Jackson ImmunoResearch;
otherwise indicated). Transfers were electro- were incubated with 0.5× blocking solution + #115-035-146, 1:10,000). All secondary anti-
phoresed at 100 V for 1 hour under ambient 0.1% Tween 20 + 0.01% SDS containing one body steps used a surface-to-volume ratio of
conditions with an ice block in the transfer or more of the following secondary antibod- 5 ml primary antibody solution per 2 ¾-inch
tank and the transfer tank surrounded by ies: IRDye 800CW–conjugated goat anti- × 3 ¼-inch membrane from a 15-well mini-
ice. rabbit (LI-COR; #926-32211, 1:20,000), IRDye gel. The membrane and secondary antibody
800CW–conjugated goat anti-mouse (LI-COR; solution were sealed in a plastic bag and in-
Immunoblotting: Membrane blocking
#926-32210, 1:20,000), IRDye 680–conju- cubated on a rotating platform for 1 hour at
After transfer, the molecular weight markers gated goat anti-rabbit (LI-COR; #926-32221, room temperature.
on the membrane were overwritten with a 1:20,000), IRDye 680LT–conjugated goat Membranes were removed from secondary
lead pencil (to provide 700-channel fluores- anti-mouse (LI-COR; #926-68020, 1:20,000), antibody solution and washed on a rocking
cence), and the membrane was blocked with and IRDye 680LT–conjugated donkey anti- platform for 4 × 5 min in ~25 ml of TBS-T.
0.5× blocking buffer: Odyssey blocking buf- chicken (LI-COR; #926-68028, 1:20,000). All To enable a fair comparison with fluores-
fer (LI-COR; #927-40000) diluted in an equal secondary antibody steps used a surface-to- cence detection, the membrane was washed
volume of PBS. Although not observed for the volume ratio of 5-ml primary antibody solu- for 5 min in ~25 ml of TBS before exposing.
Table 1. List of antibodies used and proteins or epitopes detected. BD, acetic acid, and PEG-400 (polyethylene glycol,
BD Biosciences; CST, Cell Signaling Technology; SCBT, Santa Cruz Bio- molecular weight 400). For erasing, Ponceau
technology; Thermo, Thermo Fisher Scientific; KLF4, Kruppel-like factor 4; S–stained membranes were treated with 0.1
MCL1, myeloid cell leukemia 1; PDI, protein disulfide isomerase. N NaOH for 30 s and washed with running
deionized water for 2 min.
For both total protein stains, digital images
were captured on a ChemiDoc MP gel imager
(Bio-Rad) with “Colorimetric” settings (2 × 2
camera binning). Fluorescence images were
obtained on an Odyssey infrared scanner (LI-
COR) at 169-μm resolution and 0-mm focus
offset, with a 700-channel intensity of 5.0 and
an 800-channel intensity of 8.0.
Coomassie staining and digital image
acquisition
Polyacrylamide gels were stained with 0.1%
(w/v) Coomassie blue R-250 in 40% methanol
and 10% glacial acetic acid on a rocking plat-
form for 1 hour at room temperature and then
destained for several hours with 30% metha-
nol + 10% glacial acetic acid until the back-
ground was acceptable. The stained gel was
scanned on an Odyssey infrared scanner (LI-
COR) at 169-μm resolution and 0.5-mm focus
offset for 700-channel fluorescence.
Image densitometry
Raw 16-bit TIFs (tagged image files) were
opened in ImageJ (5) and rotated to align
immunoblot bands horizontally in the win-
dow. The rectangle tool was then used to
select lanes containing the band of interest.
The width of the lane rectangle was drawn
as wide as possible without causing overlap
with bands from adjacent lanes. The height
of the lane rectangle was drawn long enough
to get a sample of the local background sur-
rounding the band of interest. Lane profiles
were plotted with the gel analysis plug-in,
Membranes were covered with an ECL solu- followed by a 5-min wash with PBS before and background was subtracted by connect-
tion composed of 1.25 mM luminol, 2 mM blocking and immunodetection as described ing the background intensity profiles to the
4-iodophenylboronic acid, and 0.0162% H2O2 above. For the SDS plus β-mercaptoethanol left (top) and the right (bottom) of the band
(16). Alternatively, membranes were covered strip, membranes were incubated with of interest by using the line tool. Last, the
with SuperSignal West Femto reagent ac- SDS plus β-mercaptoethanol solution [62.5 magic-wand tool was used to calculate the
cording to the manufacturer’s instructions mM tris (pH 6.8), 2% SDS, and 100 mM integrated area within the band profile of in-
(Thermo Fisher Scientific). Chemilumines- β-mercaptoethanol] and incubated in a dry- terest and obtain the final raw densitometry
cent exposures were captured on a Chemi- air oven at 50°C for 30 min with occasional value.
Doc MP gel imager (Bio-Rad) with “Chemi Hi agitation by hand. Stripped membranes were
Recombinant protein purification
Resolution” settings (2 × 2 camera binning). washed 3 × 5 min in ~25 ml of PBS before
Exposure times were set manually to fill the blocking and immunodetection as described Recombinant GST-ERK2 and GST-p38 were
bit depth of the CCD camera without saturat- above. prepared by glutathione affinity chromatog-
ing any binned pixels. raphy in RIPL cells (Stratagene) as described
Total protein staining after
(22).
Immunoblotting: Stripping and electrophoretic transfer
reprobing Statistical analysis
PVDF membranes were stained for total
For the low-pH glycine strip, membranes protein with MemCode Reversible Protein Serial dilutions were fit to linear or hyperbolic
were incubated with low-pH glycine buffer Stain (Thermo Scientific) according to the models by least-squares regression in IGOR
solution [1.5% glycine (pH 2.2), 0.1% SDS, manufacturer’s recommendations. Erasure Pro (WaveMetrics). The χ2 statistic from each
and 1% Tween] for 2 × 10 min at room of the MemCode stain was performed with model fit was used together with the number
temperature on a rocking platform. The the Eraser/Methanol solution for 20 min. of data points and the number of fitted
stripped membranes were washed 2 × 10 For total protein staining with Ponceau S, parameters (one for the linear model and two
min in ~25 ml of PBS before blocking and membranes were incubated with 0.1% (w/v) for the hypergeometric model) to calculate
immunodetection as described above. For Ponceau S in 5% acetic acid for 5 min and an F statistic that compares goodness of fit
the guanidinium strip, membranes were washed 2 × 5 min in 10% acetic acid, followed between models. To correct for multiple-
incubated with 6 M guanidine-HCl for 10 min by washes of 5 min in 100% methanol and 5 hypothesis testing, FDRs were calculated
at room temperature on a rocking platform, min in a 70:30:4 volume ratio of methanol, according to Benjamini and Hochberg (47).
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A neuronal antigen in the brains of

Alzheimer patients
Benjamin L. Wolozin, Alex Pruchnicki, Dennis W. Dickson, Peter Davies
A monoclonal antibody was prepared against pooled homogenates of brain tissue from
patients with Alzheimer’s disease. This antibody recognizes an antigen present in much
higher concentration in certain brain regions of Alzheimer patients than in normal brain.
The antigen appears to be a protein present in neurons involved in the formation of
neuritic plaques and neurofibrillary tangles, and in some morphologically normal neurons
in sections from Alzheimer brains. Partial purification and Western blot analysis revealed Fig. 1. Quantitation of Alz-50 reactivity in
the antigen from Alzheimer brain to be a single protein with a molecular weight of 68,000. temporal cortex of patients that had died of
Application of the same purification procedure to normal brain tissue results in the detec- Alzheimer’s disease and normal individuals.
tion of small amounts of a protein of lower molecular weight. Alzheimer’s disease cases were typical in both
clinical and neuropathologic features (3). Brains
were obtained from normal individuals dying in
A
lzheimer’s disease is a neurodegenera- gated goat antibody to mouse immunoglobu- hospital from lung or heart disease. These pa-
tive disorder characterized clinically lins. Those antibodies that showed greater tients were not demented prior to death and had
by progressive loss of intellectual func- than a 50% increase or decrease in binding no history of neurologic or psychiatric disease;
tion. This impairment of function ap- to homogenates of Alzheimer brain relative neuropathologic studies failed to reveal any sig-
pears to be correlated with numbers of to normal tissue were studied further. One of nificant pathology. The reactivity in the brains of
neuritic plaques in the neocortex and with these antibodies, Alz-50, is described below. the Alzheimer patients is estimated to be 15 to
loss of presynaptic markers of cholinergic Initial assays showed that the binding of 30 times greater than in the brains of the normal
neurons (1). Neuritic plaques are composed Alz-50 was highly selective for brain tissue subjects. Various amounts of antigen (x axis)
of degenerating axons and nerve terminals, from Alzheimer patients. Figure 1 shows that was dried onto 90-well polyvinyl plates (NUNC,
often surrounding an amyloid core and usu- 0.33 µg of temporal cortex homogenate from Germany). Nonspecific binding of protein to the
ally containing reactive glial elements (2). Alzheimer patients gave an optical density antigen was blocked by incubating the plates
Another characteristic pathologic feature only slightly lower than 10 µg of temporal with 0.01M TBS, pH 7.4, plus 5% dried milk for 1
of Alzheimer’s disease, the neurofibrillary cortex homogenate from normal patients. hour. Alz-50 was diluted 1:5 in blocking solution
tangle, is an intraneuronal mass composed From these data we conclude that the anti- and incubated overnight at 4°C. Unbound anti-
of normal intermediate filaments and paired gen is elevated 15 to 30 times in the temporal body was removed by washing five times with
helical filaments (PHF) with unusual proper- cortices of the Alzheimer patients. Alz-50 re- 0.02% Tween-TBS. Peroxidase-coupled goat an-
ties (3). activity was similarly elevated in the nucleus tibody to mouse immunoglobulins (Kirkegaard
In studying the topographic distribution basalis and hippocampus. These areas, cor- & Perry) was diluted 1:100 in blocking solution,
of plaques and tangles in the brains of Al- tex, nucleus basalis, and hippocampus, are added to the plates, and incubated for 1 hour
zheimer patients, we noted that the lesions all known to contain neuritic plaques and at room temperature. After five washes with
occur with high frequency in regions receiv- neurofibrillary tangles in brains of patients 0.02% Tween-TBS, reactivity was visualized with
ing cholinergic innervation from the ventral with Alzheimer’s disease. Brain areas less af- 2,2’-azino-di’-3-ethyl-benzthiazoline solution
forebrain (4). This cholinergic cell group fected by the disease, such as caudate, thala- (ABTS) (Kirkegaard & Perry). Results are ex-
appears to be extremely vulnerable to the mus, or cerebellum demonstrated little or no pressed as means, with standard deviations indi-
disease process, and evidence that cholin- reactivity. cated. Numbers of cases were ten for Alzheimer
ergic nerve terminals participate in plaque The immunocytochemistry of Alz-50 on and six for normal.
formation has been presented (5). To better Formalin-fixed tissue was dramatic and was
define the relation between ventral forebrain consistent with the hypothesis that Alz-50 50, and reactivity was visualized with the use
cholinergic neurons and the lesions of the was highly selective for neuronal compo- of peroxidase conjugated goat antibody to
Alzheimer brain, we have prepared mon- nents in Alzheimer brain (Fig. 2A). Many mouse immunoglobulins. 4-Chloronaphthol
odonal antibodies to homogenates of ventral neurons in the pyramidal layer of the hippo- was used to visualize the peroxidase reac-
forebrain tissue taken at autopsy from four campus were stained. The antigen was pres- tion; this compound, a peroxidase substrate,
patients with Alzheimer’s disease. The re- ent in cell bodies and in neurites. Figure 2C yields a product that precipitates in aqueous
sulting antibodies were screened on the basis shows that plaques were strongly stained by solution but is soluble in organic solvents.
of their ability to differentiate brain tissue the antibody. The staining was confined to The tissue section was photographed and the
from patients with Alzheimer’s disease and the neuritic meshwork present in plaques. 4-chloronaphthol was removed by dehydra-
from normal subjects in both immunochemi- Darkly stained neurons and plaques were tion and xylene treatment. Finally, plaques
cal and immunocytochemical procedures. found throughout Alzheimer hippocampus and tangles were stained with thioliavine S,
Antibodies were initially assayed ac- and cortex. By contrast, there was virtually a sensitive histologic reagent for the demon-
cording to their ability to bind to brain ho- no staining of normal brain (Fig. 2B). This stration of these lesions, and the section was
mogenate that had been immobilized onto pattern of specificity was observed in a total photographed again.
polyvinyl plates (1, µg per 50-mm diameter of eight brains from Alzheimer patients and Comparison of the staining patterns (Fig.
well) by drying at 37°C for 1 hour. Antibody five brains from normal subjects. 3) revealed that many neurons were stained
binding was detected with peroxidase-conju- To determine the relation between Alz-50 both by the antibody and by thioflavine
staining of neurons and the presence of neu- S. However, several neurons were darkly
rofibrillary tangles we used a double staining stained by the antibody and did not appear to
Departments of Pathology and Neuroscience, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, technique. Vibratome sections of Formalin- contain neurofibrillary tangles. A small frac-
Bronx, NY 10461 fixed Alzheimer tissue were reacted with Alz- tion of neurons that contained tangles and
22 Originally published 2 May 1986 in SCIENCE sciencemag.org SCIENCE

were thioflavine-positive were in native form on a Sepharose

not positive for Alz-50 (Fig. 3, A 6B column. Temporal cortex
and B). The staining of plaques homogenate from Alzheimer
by Alz-50 was also studied by patients or normal subjects was
this method. All plaques bound centrifuged at 27,200g for 20
both Alz-50 and thioflavine. minutes, and the supernatant
In addition, these results con- was run through the Sepharose
firmed the neuritic nature of 6B column. An immunoreac-
the antibody staining; staining tivity profile was obtained by
was present in the neuritic pe- diluting each fraction 1:100 in
riphery of the plaques but ab- water, drying 50 µl of diluted
sent in the amyloid core. fraction onto polyvinyl plates,
Simple biochemical experi- and assaying by ELISA. The
ments suggest that the Alz-50 profile from Alzheimer brain
antigen is distinct from PHF, Fig. 2. (A) Immunocytochemistry of Alz-50 staining in the pyramidal revealed a single immunoreac-
the major tangle component. layer of hippocampus from a brain affected by Alzheimer's disease. tive peak at the void volume.
Unlike PHF, the Alz-50 anti- Vibratome sections (40 µm) were cut from Formalin-fixed brain. The tis- By contrast, no immunoreac-
gen is largely soluble in 0.01M sue was washed twice in TBS, incubated for 30 minutes in 0.25% Triton X- tivity was seen in the column
tris-buffered saline (TBS) and 100-TBS, washed once in TBS, and incubated for 30 minutes in dried milk fractionation of normal brain
completely soluble in TBS con- (blocking solution) for 1 hour at room temperature to prevent nonspecific at this dilution. At a lesser di-
taining 5% sodium dodecyl binding of antibody. Alz-50 was diluted 1:5 in blocking solution and incu- lution, normal brain immuno-
sulfate (TBS-SDS) (6). Solubil- bated with the tissue sections overnight at 4°C. Unbound antibody was reactivity was seen at the void
ity was tested by vortexing removed by washing in TBS. Peroxidase coupled goat antibody to mouse volume. This result supports
Alzheimer cortex homogenate immunoglobulin G was diluted 1:100 in blocking solution and incubated with the quantitative data shown in
for 2 minutes in TBS or TBS- the tissue sections for 1 hour. Antibody was visualized by incubating the sec- Fig. 1 demonstrating 15 to 30
SDS. The homogenate was then tions for 8 minutes in a 0.1M tris solution, pH 7.4, containing 0.45 mg/ml times as much inimunoreactiv-
centrifuged at 10,000g for 10 of diaminobenzidine and 0.44 mM hydrogen peroxide. The tissue was then ity in the Alzheimer brain as in
minutes. Supernatant and pel- washed in TBS, dehydrated, and mounted. Magnification x 12.5. (B) Alz-50 the normal brain.
let were separated, and the staining in hippocampus from a normal brain. (C) A plaque from frontal cor- Samples of supernatant from
pellet was washed twice by tex of a brain affected with Alzheimer’s disease. Magnification x25. Alzheimer and normal cor-
homogenizing and centrifug- tex were then studied by the
ing as above. The supernatant Western blot technique (Fig. 4).
and pellet were homogenized The major band in Fig. 4, lane
in water and various amounts A, has an apparent molecular
of each sample (10, 3, 1 µg per weight of 68,000 (68K). In Fig.
50, µl) were dried onto poly- 4, lane B, the reactivity from
vinyl plates. The presence of the normal void volume frac-
antigen was determined by an tion was brought out by load-
enzyme-linked immunosorbent ing onto the polyacrylamide gel
assay (ELISA). PHF reactivity three times as much protein as
was monitored by means of was used for the Western blot
an antibody to PHF, antibody of the Alzheimer void volume
704.1 (10). Alz-50 reactivity was Fig. 3. (A) Immunocytochemistry of Alz-50 in hippocampus from a brain fraction. The major band has
found in the TBS supernatant affected by Alzheimer’s disease. Arrows point to neurons that react with an apparent molecular weight
and was quantitatively recov- Alz-50 (A) but do not stain with thioflavine S (B). For details on the immuno- of 59K. A doublet at 245K is
ered in the TBS-SDS superna- cytochemistry see legend to Fig. 1. The peroxidase reaction was developed also present.
tant, whereas PHF reactivity by using 0.2 mg/ml of 4-chloronaphthol instead of diaminobenzidine (as in These results show that in
remained in the pellet after Fig. 2). Microscopy was performed by using phosphate buffered saline (PBS)- TBS the antigen recognized
TBS-SDS extraction. Thus, Alz- glycerol instead of dehydration. Magnification x 12.5. (B) Thioflavine S histo- by Alz-50 is either aggregated
50 immunoreactivity is soluble chemistry of the same section from (A). Note the absence of thioflavine S or is part of a large complex.
and segregates away from PHF staining of neurons (arrows). To remove the 4-chloronaphthol the section was When dissociated, the epitope
immunoreactivity. dehydrated, whereupon the 4-chloronaphthol dissolved in the xylene, and occurred as a single 68K pro-
Enzyme experiments con- then subsequently rehydrated. The section was then incubated in a 0.01% tein that was distinctly differ-
firmed this result. Before we thioflavine S solution in Formalin and rinsed by dipping three times in 80% ent from the 59K antigen from
dried the Alzheimer brain ethanol (fluorescence microscopy; magnification x 12.5.) normal brain. Several relevant
homogenates onto polyvinyl proteins have subunit molecu-
plates, we treated them for 0, lar weights in the 68K range: in
20, or 60 minutes with trypsin or alkaline lated, although it is of interest because many the cytoskeletal family there are neurofila-
phosphatase. An ELISA was used to measure antibodies to neurofilament epitopes that ment and tau proteins (7), in the cholinergic
the sensitivity of the Alz-50 epitopes to the identify neurofibrillary tangles fail to react family there is choline acetyltransferase (8).
treatment. Unlike the PHF, the Alz-50 epit- after phosphatase treatment. In addition, The Alz-50 antigen is therefore unlikely to be
ope was highly sensitive to trypsin. This sup- this result suggests that phosphorylation of neurofilament, tau protein, or choline acetyl-
ports the hypothesis that the Alz-50 antigen the epitope does not account for the ability transferase, because none of these proteins
is not PHF, and also demonstrates that the of the antibody to distinguish between Al- seems to be elevated in concentrations 15 to
antigen is a protein. The Alz-50 epitope is not zheimer and normal brain. 30 times in the brains of Alzheimer patients.
phosphatase sensitive; this does not prove A Western blot of the Alz-50 antigen was Further, if the 68K Alzheimer type pro-
that the Alz-50 antigen is not phosphory- obtained by first purifying the Alz-50 antigen tein is related to the 59K protein, then the
Fig. 4. Western blot of (A) Alzheimer and REFERENCES AND NOTES

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used instead of peroxidase-coupled antibody. 215, 1243 (1982).
Color development was achieved by means of 7. F. C. Chiu, W. T. Norton, K. L. Fields, J.
BCIP/NBT (Kirkegaard & Perry). Omission of Neurochem. 37, 147 (1981).
the incubation with Alz-50 completely abol- 8. A. J. Levey, D. B. Rye, B. H. Wainer, ibid. 39,
ished staining. 1652 (1982).
9. D. W. Dickson, Y. Kress, A. Crowe, S.-H. Yen,
Am. J. Pathol. 120, 292 (1985).
Alz-50 antigen is unlikely to be neurofila- minals in plaques and in most neurons with ACKNOWLEDGMENTS
ment, tau protein, or choline acetyltrars- tangles. The surprising finding is that Alz- Supported by NIH training grant T32 GM7288
ferase. The relation among the proteins 50 immunoreactivity appears to precede the from the National Institute of General Medical
detected is questionable: the relatively large deposition of neurofibrils and PHFs to form Sciences, The Mcknight Foundation, The
amounts of samples of normal brain tissue tangles. Thus Alz-50 may recognize a precur- Joyce Mertz-Gilmore Foundation, and the
needed to detect any reactivity raises doubts sor to tangle formation. Alternatively, Alz-50 Commonwealth Fund. We thank S.-H. Yen for
about the specificity of the antibody binding. immunoreactive neurons may be affected antibody 704.1 to PHF and M. Scharff and E.
Some monoclonal antibodies to tangles react by Alzheimer pathophysiology but may not Fischberg for advice and assistance. We also
with proteins in the 59K to 68K range (9), but form classical tangles. The biochemical data acknowledge use of the hybridoma facility of the
none of these show quantitative differences showing that Alz-50 is not PHF antigen is cancer center (CA 13330) at the Albert Einstein
between normal and Alzheimer brain. The consistent with this discordance between College of Medicine.
points raised above suggest that the epitope the presence of Alz-50 and the presence of
recognized by Alz-50 is a novel antigen. neurofibrillary tangle. The function and 30 October 1985; accepted 18 February 1986
Our results demonstrate that Alz-50 rec- identity of the proteins that are recognized
ognizes a protein present in neuronal ter- by Alz-50 remains to be elucidated.
Serological analysis of a subgroup of male controls. Sera from the same patients
were also tested by the enzyme-linked
human T-lymphotropic retroviruses

immunosorbent assay (ELISA) with purified
HTLV-III as part of a larger, systematic
serologic study of the prevalence of
(HTLV-III) associated with AIDS antibodies to HTLV-III in AIDS and pre-

AIDS patients (19).
Representative results are shown in Fig.
Jörg Schüpbach,1 Mikulas Popovic,1 Raymond V. Gilden,2 Matthew A. Gonda,2 1. Sera from patients with AIDS or pre-
M. G. Sarngadharan,3 and Robert C. Gallo1 AIDS, and from some homosexuals and
heroin-addicts, recognized a number of
The two main subgroups of the family of human T-lymphotropic retroviruses (HTLV) that specific antigens not detected by sera from
have previously been characterized are known as HTLV-I and HTLV-II. Both are associ- heterosexual subjects. The most prominent
ated with certain human leukemias and lymphomas. Cell surface antigens (p61 and p65) reactions were with antigens of the following
encoded by HTLV-I are frequently recognized, at low titers, by antibodies in the serum of molecular weights: 65,000, 60,000, 55,000,
patients with acquired immunodeficiency syndrome (AIDS) or with signs or symptoms 41,000, and 24,000. Antigens with molecular
that precede AIDS (pre-AIDS). This suggests an involvement of HTLV in these disor- weights of approximately 88,000, 80,000,
ders. Another subgroup of HTLV, designated HTLV-III, has now been isolated from many 39,000, 32,000, 28,000, and 21,000 gave less
patients with AIDS and pre-AIDS. In the studies described in this report, virus-associated prominent reactions. The reaction with the
antigens in T-cell clones permanently producing HTLV-III were subjected to biochemical antigen of 55,000 (p55) only occurred in
and immunological analyses. Antigens of HTLV-III, specifically detected by antibodies sera that also recognized p24, suggesting a
in serum from AIDS or pre-AIDS patients and revealed by the Western blot technique, relationship between the two antigens.
are similar in size to those found in other subgroups of HTLV. They include at least three The specificity of these reactions was
serologically unrelated antigenic groups, one of which is associated with group-specific studied by comparing lysates of H4/HTLV-
antigens (p55 and p24) and another with envelope-related (p65) proteins, while the anti- III and HI7/HTLV-III with lysates of the
gens in the third group are of unknown affiliation. The data show that HTLV-III is clearly same cell clones, H4 and H17, before viral
distinguishable from HTLV-I and HTLV-II but is also significantly related to both viruses. infection (Fig. 2A). No antigen from the
HTLV-III is thus a true member of the HTLV family. uninfected clones reacted with the sera,
with the exception of a protein with a
M
embers of the family of human lym- of blood transfusion recipients who later molecular weight of 80,000 in HI7 which
photropic retroviruses (HTLV) have developed AIDS and of their blood donors bound antibodies from all of the human
the following features in common: a have revealed the presence, in the blood of serum samples tested (see Fig. 1B) but not
pronounced tropism for OKT4+ lym- the donors, of antibodies to a retrovirus of from rabbit or goat serum. Antigens newly
phocytes (1), a reverse transcriptase the HTLV family (14). These findings suggest expressed after viral infection and recognized
(RT) with a high molecular weight (100,000) an involvement of viruses of the HTLV by the human serum used for this analysis
and a preference for Mg2+ as the divalent cat- family in the cause of AIDS and pre-AIDS. included p65, p55, p41, p39, p32, and p24.
ion for optimal enzymatic activity (2, 3), and An involvement of HTLV-I alone appeared A large protein with a molecular weight
the capacity to inhibit T cell function (4) or, doubtful, however, because antibody titers of approximately 130,000 and a protein of
in some cases, kill T cells (5). Many HTLV to gp6l of HTLV-I in these patients are 48,000 were also detected. With this serum,
also have the capacity to transform infected generally very low and antibodies to the p55 consistently appeared as a doublet of
T cells (1). The two major subgroups that have structural proteins of HTLV, notably p24 bands of similar intensity. With normal
been characterized (6) are HTLV-I, which is and p19 (15), are not detectable in most AIDS human serum, none of the antigens was
causatively linked to certain adult T-cell ma- patients (16). Instead, it seemed likely that detected (not shown). These results show
lignancies (7), and HTLV-II, which was first another member of the HTLV family might clearly that the antigens detected after virus
identified in a patient with hairy cell leuke- be involved in the etiology of AIDS. Here we infection are either virus-coded proteins or
mia (8). describe our studies of a group of cytopathic cellular antigens specifically induced by the
Viruses of the HTLV family have been yiruses (collectively designated HTLV-III) infection.
detected in some patients with the acquired isolated from patients with AIDS or pre- The antigens of H4/HTLV-III were also
immunodeficiency syndrome (AIDS) (9) AIDS. Isolation of these viruses was achieved compared with antigens from virus purified
or with pre-AIDS, a condition frequently by means of a novel system permitting the from the culture fluids of H4/HTLV-III (Fig.
progressing to AIDS (10). A high proportion continuous growth of T-cell clones infected 2B). Extensive accumulation of p24 and p41
of patients with AIDS or pre-AIDS, as well as with the cytopathic types of HTLV found in [see (20)] occurred in the virus preparation
a significant number of hemophiliacs, have these disorders (17). We show that antigens (Fig. 2B, panels I and II). Protein stains
antibodies in their serum that recognize associated with human cells infected by showed that these molecules are the major
a cell surface glycoprotein (gp6l) that is HTLV-IlI are specifically recognized by components of the virus preparation (19).
present on certain human T cells infected antibodies in serum from AIDS and pre- P24 and p41 may therefore be considered
with HTLV-I (11). Gp6l and p65, a slightly AIDS patients, and present a preliminary viral structural proteins. Furthermore,
larger protein that is a homolog of gp6l and biochemical and immunological analysis of an antigen with a molecular weight of
occurs in another cell line producing HTLV-I, these antigens. approximately 110,000 was detected in the
were subsequently shown to be related to Lysates of two immortalized and infected virus preparation but was below limit of
the HTLV viral glycoprotein (12, 13). Studies human T-cell clones, H4/HTLV-III and H17/ detection in the cells. Also, p39 [see (20)]
HTLV-III (17), were tested with samples of was present in the virus preparation. It is
1
Laboratory of Tumor Cell Biology, National Cancer Institute, human serum in a strip radioimmunoassay interesting that p24 in the virus preparation
Bethesda, Maryland 20205. (RIA) based on the Western blot techniq1ue consistently appeared as a doublet (p24/
2
Program Resources, Inc. NCI-Frederick Cancer Research Facility,
(18). The sera were from patients with p23), whereas in the cells it appeared as
Frederick, Maryland 21701.
3
Department of Cell Biology Litton Bionetics, Inc., Kensington, AIDS or pre-AIDS, from contacts of such p24 alone. The significance of this is under
Maryland 20895. patients, and from homo- or heterosexual investigation. P55 was not detected in the
SCIENCE sciencemag.org Originally published 4 May 1984 in SCIENCE 25

Fig. 1. Serologic detection of antigens in HTLV-III producer cell Fig. 2. (A) Specificity of the antigens recognized. Lysates of
clones. Strip RIA were performed with human serum as described cloned cells before and after infection with HTLV-III were analyzed
elsewhere in detail (21). Briefly, lysates of HTLV-IlI producer cell by the Western blot technique (18) with a 1:500 dilution of the
clones were subjected to electrophoresis under reducing conditions serum shown in Fig. 1B, lane e. (Lane a) The HI7 clone before and
on preparative sodium dodecyl sulfate (SDS)-polyacrylamide slab (lane b) the same clone after infection (HI7/HTLV-IlI); (lane c) the
gels, and electroblotted to nitrocellulose sheets (18). The sheets were H4 clone before and (lane d) the same clone after infection (H4/
cut into strips. These were incubated with human serum diluted 1:100. HTLV-III). All reactive antigens are virus-related with the exception
After three thorough washings, bound antibodies of immunoglobulin of that with a molecular weight of 80,000 in HI7 cells; this antigen
G (lgG) and immunoglobulin M (lgM) classes were made visible with binds antibodies from all human sera investigated. Normal human
radiolabeled, affinity-purified goat antiserum to human IgG and IgM serum did not bind to any of the virus-related bands (not shown).
(H-chain specific) and autoradiography. (A) Analysis with H4/HTLV- (B) Comparison of antigens in (lanes a) cells and (lanes b) virus.
III cells. (Lanes a, d, and g) U.S. patients with AIDS; (lane b) a French Lysates of H4/HTLV-III (250, µg per lane) or virus purified from
heterosexual male who developed AIDS after receiving a blood the cell culture fluids (19) (5 µg per lane) were analyzed with
transfusion in Haiti (24); (lane c) an AIDS patient from Switzerland; 1:500 dilutions of human sera. (Panel I) Same serum as in Fig.
(lane e) a normal heterosexual control; (lane f) a French pre-AIDS 2A; (panel II) serum of a Swiss male homosexual with fatigue and
patient (24); (lane h) a Swiss heterosexual drug addict; (lane i) a generalized lymphadenopathy (pre-AIDS); (panel III) serum from
normal homosexual control. (B) Analysis with H17/HTLV-IlI cells. same AIDS patient as in Fig. IB, lane d. An antigen with a molecular
(Lane a) An infant with AIDS whose mother is a prostitute; sera from weight of 110,000 and p41, p39, and p24 are enriched in the virus
both are highly positive for antibodies to the HTLV membrane antigen preparation [see (20)]. The serum in panel III recognized a subset
(11, 25) and in our ELISA with disrupted HTLV-III (19); (lane b) same of the antigens recognized by the sera used in panels I and II.
serum as in (A), lane d; (lane c) normal heterosexual control; (lane
d) another Swiss AIDS patient; (lane e) a Swiss heterosexual male
intravenous drug abuser with generalized lymphadenopathy and
thrombocytopenic purpura (pre-AIDS).
Fig. 3. Relation between HTLV-

III and HTLV-II. Serum of an
AIDS patient at a dilution of 1:
500 was tested in a competition
RIA on strips (20) prepared with
H4/HTLV-III cells. (Lane a) The
human serum was added directly
to the strip (uncompeted control);
(lanes b to e) the serum was first
absorbed for 3 hours at 37°C with
1 mg of cellular extract. In (b) the
absorption was with uninfected
H4 cells (not producing virus); in
(c) the absorption was with H4/
HTLV-IlI cells producing HTLV-
III (positive control); in (d) the
absorption was with C3/44 cells
(26) producing HTLV-II; in (e) the
absorption was with HUT 102
cells producing HTLV-I (2).
Fig. 4. Electron microscopy of thin sections of cells producing HTLV-I, -II, and
-III. (Top) HUT 102 cells producing HTLV-I (2). (Middle) Cells from an AIDS patient
(J.P.) producing HTLV-II (24). (Bottom) Cells from a patient [described in (27)]
with pre-AIDS, producing HTLV-III. (Panels a) Virus particles budding from the cell
membrane. (Panels b) Free particles have separated from the membrane. (Panels c)
Free particles sectioned in a different plane, Note the dense, cylindrical core region
of HTLV-III.
virus; however, the intensity of the p55 band nucleic acid data clearly indicate that HTLV- 15. V. S. Kalyanaraman, M. G. Sarngadharan, P.
in the cells (Fig. 2B, lanes a) appeared to III is a true member of the HTLV family and A. Bunn, J. D. Minna, R. C. Gallo, Nature
correlate with the intensity of p24/p23 in that it is more closely related to HTLV-II (London) 294, 271 (1981); V. S. Kalyanaraman,
the virus preparation (Fig. 2B, lanes b), thus than to HTLV-I. M. Jarvis-Morar, M. G. Sarngadharan, R. C.
again suggesting a relation between these Gallo, Virology 132, 61 (1984).
antigens. The p55 is probably a precursor of REFERENCES AND NOTES 16. M. Robert-Gurotr et al.; in Cancer Cells, vol. 3,
p24, since a group-specific antigen of similar 1. M. Popovic, P. S. Sarin. M. Roben-Guroff, V. Human T-Cell Leukemia Viruses, R. C. Gallo
size (Pr 54gag ) in HTLV-I-infected cells is the S. Kalyanaraman, D. Mann, J. Minowada, R. C. and M. Essex, Eds. (Cold Spring Harbor
precursor of p24 and the other gag-coded Gallo, Science 219, 856 (1983); P. D. Markham, Laboratory, Cold Spring Harbor, N.Y., in press),
proteins (21). Occasionally an additional S. Z. Salahuddin, V. S. Kalyanaraman, M. 17. M. Popovic, M. G, Sarngadharan. E. Read, R.
set of antigens was recognized by a serum Popovic, P. Sarin, R. C. Gallo, Int. J. Cancer 31, C. Gallo, Science 224, 497 (1984).
(Fig. 2B, panel III) but their relation to the 413 (1983); S. Z. Salahuddin. P. D. Markham, F. 18. H. Towbin, T. Staehelin, J. Gordon, Proc. Natl.
antigens described above is unclear. Wong-Staal, G. Franchini, V. S. Kalyanaraman, Acad. Sci. U.S.A. 76, 4350 (1979).
Thus we have shown that viral or virus- R. C. Gallo, Virology 129, 51 (1983). 19. M. G. Sarngadharan, M. Popovic, L. Bruch, J.
induced antigens in cloned human T cells 2. B. J. Poiesz, F. W. Ruscetti, A. F. Gazdar, P. Schüpbach, R. C. Gallo, Science 224, 506
infected with HTLV-III are specifically A. Bunn, J. D. Minna. R. C. Gallo, Proc. Natl. (1984).
recognized by antibodies in the serum Acad. Sci. U.S.A. 77, 7415 (1980). 20. Although in Fig. 2B the p41 in the virus
of patients with AIDS or pre-AIDS. The 3. H. M. Rho, B. Poiesz, F. W. Ruscetti. R. C. preparation appears to be larger than the p41
detection of p65 by many of the serum Gallo, Virology 112, 355 (1981); M. Seiki, S. in cells, the two molecules are of the same
samples is of special interest. We have tested Hattori, Y. Hirayama, M. Yoshida, Proc. Natl. size. During application of the lysates to the
these sera on strips prepared from lysates Acad. Sci. U.S.A. 80, 3618 (1983). gel in another experiment, a small amount of
of cells producing HTLV-I or -II. Some of 4. M. Popovic et al.. in preparation. cellular lysate was spilled into the neighboring
these cells produce a p65 that has been 5. H. Mitsuya, H. G. Guo, M. Megson, C. O. lanes and the cellular p41 moved with the
shown (13) to be coded for by the env gene Trainor, M. S. Reitz, S. Broder, Science 223, same velocity as the viral p41. A connecting
of HTLV-I and to be the homolog of the gp61 1293 (1984). band was thus formed between the p41 in
described by others (11, 12). Many of the sera 6. For a brief review, see M. G. Sarngadharan et the lane containing the cells and the p41 in
recognizing p65 in HTLV-III–infected cells al., in Human Carcinogenesis, C. C. Harris and the lane with the virus. The same situation
also recognized, though somewhat faintly, H. H. Autrup, Eds. (Academic Press, New occurred with p39 in cells and virus.
p65 in cells producing HTLV-I or -II, and York, 1983), p. 679. 21. J. Schüpbach, V. S. Kalyanaraman, M. G.
some of them also recognized gag-related 7. V. S. Kalyanaraman, M. G. Sarngadharan. Y. Sarngadharan, R. C. Gallo, in preparation.
antigens (data not shown). Nakao, Y. Ito. T. Aoki, R. C. Gallo, Proc. Natl. 22. M. G. Sarngadharan et al., in preparation.
In addition, the reaction of some human Acad. Sci. U.S.A. 79, 1653 (1982); M. Robert- 23. S. Arya et al., in preparation.
sera with virus-related antigens of HTLV-III– Guroff, Y. Nakao, K. Notake, Y. Ito, A. H. 24. J. B. Brunet et al., Lancet 1983-I, 700 (1983).
infected cells could be partially inhibited by Sliski, R. C. Gallo, Science 215, 925 (1982); Y. 25. M. Essex, personal communication.
large amounts of extracts of cells producing Hinuma et al., Int. J. Cancer 29, 631 (1982); 26. M. Popovic, V. S. Kalyanaraman, D. L. Mann,
HTLV-II (Fig. 3). When a human serum not W. A. Blattner et al., ibid. 30, 257 (1982); E. Richardson, P. S. Sarin, R. C. Gallo, in
recognizing p65 was used, the antigens for J. Schüpbach, V. S. Kalyanaraman, M. G. Cancer Cells, vol. 3, Human T-Cell Leukemia
which there was competition included p55, Sarngadharan, Y. Nakao, R. C. Gallo, ibid. 32, Viruses, R. C. Gallo and M. Essex, Eds. (Cold
p48, p41, p39, and p24. These results were 583 (1983); J. Schüpbach, V. S. Kalyanaraman, Spring Harbor Laboratory, Cold Spring Harbor,
confirmed by the demonstration that a rabbit M. G. Sarngadharan, W. A. Blattner, R. C. Gallo, N.Y., in press).
antiserum raised against purified HTLV-III Cancer Res. 43, 886 (1983). 27. R. C. Gallo et al., Science 224. 500 (1984).
showed some reactivity with antigens of 8. V. S. Kalyanaraman et aI., Science 218, 571
HTLV-II and, to a lesser extent, with HTLV-I. (1982); E. P. Gelmann et al., Proc. Natl. Acad. ACKNOWLEDGMENTS
In contrast, antiserum to HTLV-II recognized Sci. U.S.A. 81, 993 (1984). We thank J. Ahmad for technical assistance and
both HTLV-I and -III antigens, and an 9. R. C. Gallo et al., Science 220, 865 (1983); E. P. R. Lüthy and M. Vogt, Division of Infectious
antiserum to HTLV-I reacted well with Gelmann et al., ibid., p. 862. Diseases, Department of Medicine, University
HTLV-II, but only faintly with HTLV-III (22). 10. F. Barré-Sinoussi et al., ibid., p. 868. Hospital, Zurich, and O. Haller, Institute for
Moreover, nucleotide sequences of HTLV-III 11. M. Essex et al., ibid., p. 859; M. Essex et al., Immunology and Virology, University of Zu-
have been found to be related to HTLV-I and ibid. 221, 1061 (1983). rich, Zurich, Switzerland, for making some sera
-II (23). Although the morphology of HTLV- 12. T. H. Lee, J. E. Coligan, T. Homma, M. F. from AIDS and pre-AIDS patients available and
III particles appears to be somewhat different McLane, N. Tachibana, M. Essex, Proc. Natl. for providing clinical information. J .S. is a Fogarty
from the morphology of HTLV-I and -II (Fig. Acad. Sci. U.S.A., in press. International Fellow of the National Cancer Institute.
4), and although some differences are also 13. J. Schüpbach, M. G. Sarngadharan, R. C. Gallo,
found in the protein patterns of purified virus Science, in press. 30 March 1984; accepted 19 April 1984
preparations (19), these immunological and 14. H. W. Jaffe et al., Science 223, 1309 (1984).
Cell surface P-glycoprotein associated ciated with the multidrug resistance pheno-
type observed in other cell systems.
with multidrug resistance in

Each of the different mammalian cell lines
examined in this study (Table 1) was originally
selected for resistance to a specific drug, and
mammalian cell lines in each case a multidrug resistance pheno-

type typified by cross resistance to unrelated
compounds was observed. Such a phenotype
Norbert Kartner,1 John R. Riordan,2 and Victor Ling3 appears to reflect a membrane-associated
alteration (6, 24, 26). We therefore prepared
The plasma membranes of hamster, mouse, and human tumor cell lines that display membranes from these cell lines component
multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. could not be detected by for analysis of their
In every case, increased expression of a 170,000-dalton surface antigen was found to be components by sodium dodecyl sulfate-poly-
correlated with multidrug resistance. This membrane component is of identical molecu- acrylamide gel electrophoresis (SDS-PAGE)
lar size and shares some immunogenic homology with the previously characterized (22). For CHR CHO cells (lanes b to d in Fig.
P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have 1A), a protein having a molecular size of about
application to cancer therapy. 170,000 daltons appeared in the plasma mem-
brane with a staining intensity proportional to
the degree of resistance to the drug. As previ-
S
election of variants in mammalian cells Studies in model systems indicate that ously characterized, this band is referred to as
that are resistant to specific drugs, such multidrug resistance results from a reduced the P-glycoprotein (20, 22). Similarly stained
as Vinca alkaloids, maytansine, colchi- cellular accumulation of the drugs involved bands of approximately the same molecular
cine, anthracyclines, actinomycin D, (5–19), and changes in the plasma membrane size are seen in the other drug resistant cell
or bleomycin, is often accompanied by have been observed (17–23). In the well-char- lines that we examined (lanes e, h, j, and l in
expression of a complex phenotype of cross acterized colchicine-resistant (CHR) Chinese Fig. 1A). Such a component could not be de-
resistance to various unrelated drugs (1–14). hamster ovary (CHO) system, for example, tected by protein staining in the correspond-
This characteristic is referred to as the mul- genetic analyses involving cell-cell hybrids, ing drug-sensitive parental cells (lanes a, g, i,
tidrug resistance phenotype. The generation drug-sensitive revertants, and DNA-medi- and k) or drug-sensitive revertant (lane f ).
of such variants in tumor cells may be an im- ated transformants of the CHR phenogenesis As a means of further characterizing the
portant mechanism by which neoplasms be- type indicate that multidrug resistance, col- relatedness of the 170,000-dalton membrane
come resistant to treatment by combination chicine resistance, and reduced drug accu- components observed in these drug-resistant
chemotherapy. mulation are the result of the same genetic lines, an antiserum to plasma membrane ves-
alteration (21, 24, 25). Moreover, the expres- icles of the highly colchicine-resistant CHO
sion of a 170,000-dalton plasma membrane line CHRC5 was prepared. The specificity of
1
Ontario Cancer Institute, Princess Margaret Hospital, and
glycoprotein (P-glycoprotein) is invariably this antiserum for the P-glycoprotein was im-
Department of Medical Biophysics, University of Toronto, Toronto, associated with this pleiotropic phenotype proved by cross absorption with immobilized,
Ontario, M4X 1K9, Canada. (20–22, 24). The degree of drug resistance is detergent-solubilized plasma membrane
2
Research Institute, Hospital for Sick Children, and Departments correlated approximately with the amount of proteins of the parental drug-sensitive line
of Biochemistry and Clinical Biochemistry, University of Toronto,
Toronto, Ontario, M5G 1X8.
P-glycoprotein present (20, 22). The objective (21, 27). The absorbed antiserum was then
3
Ontario Cancer Institute, Princess Margaret Hospital, and of the present study is to determine whether used to examine the membrane components
Department of Medical Biophysics, University of Toronto. or not P-glycoprotein expression is also asso- of the lines shown in Fig. lA. Each of the
Table 1. Description of cell lines. Cell line CHRA3 was selected from AUXB1; CHRB3 was selected from CHRA3 in a second step; and CHRC5 was selected
from CHRB3 in a third step (6). DNRR51 was selected from AUXBI in two steps (27). 110 is a revertant cell line, selected in a single step from CHRC5 (24).
Abbreviations for drugs are: ACR, acriflavine; ADR, adriamycin; AMD, actinomycin D; CCH, colchicine; CMD, colcemid; CYB, cytochalasin B; DNR, daunoru-
bicin; EME, emetine; ERY erythromycin; ETB, ethidium bromide; GRD, gramicidin; PRO, proflavin; PUR, puromycin; VCR, vincristine; and VLB, vinblastine.
Relative resistance was calculated as the ratio of drug concentration tolerated by the resistant cells to that tolerated equally by the sensitive parent cell.
Tolerance was assessed by relative growth rates except for cell lines 110 and ECHR, whose colony-forming abilities in the presence of drug were compared
with the appropriate parental lines. Since cross resistance is shown only for drugs that have been tested and the results reported, absence of a drug from
the column on cross resistance does not imply lack of cross resistance to that drug. The drugs are listed in order of decreasing relative cross resistance.
28 Originally published 23 September 1983 in SCIENCE

Fig. 1. Electrophoretic and immunochemical

analysis of plasma membrane components.
Cell culture, plasma membrane isolation, and
electrophoresis were carried out as described
previously (21, 22, 27). (A) Gels were loaded
with 10 µg of membrane protein per lane. They
were overstained by the silver staining method
of Switzer et al. (34) and were then reduced to
acceptable intensity by soaking in a solution of
0.03M ferric ammonium sulfate in 0. 18M sulfu-
ric acid diluted 1:10, followed by a final rinse in
0.3M sodium carbonate and thorough washing
in water. Molecular size standards (Bio-Rad)
are shown in the first lane. These consist of 0.1
µg each of myosin, (200,000), β-galactosidase
(116,250), phosphorylase b (92,500), bovine
serum albumin (66,200), ovalbumin (45,500),
carbonic anhydrase (31,000), soybean tryp-
sin inhibitor (21,500), and lysozyme (14,400).
(Lane a) Drug-sensitive parent CHO cell,
AUXB1; (lane b) colchicine (0.10 µg/ml)-resistant
CHO cell CHRA3; (lane c) colchicine (3.0 µg/ml)-
resistant CHO cell CHRB3; (lane d) colchicine
(10 µg/ml)-resistant CHO cell CHRC5; (lane e)
daunorubicin-resistant CHO cell DNRR51; (lane
f) colchicine-revertant CHO cell I10 (derived
from CHRC5); (lane g) sensitive parent SV40-
transformed Syrian hamster cell Cl2TSV5S; (lane
h) actinomycin D-resistant SV40-transformed
Syrian hamster cell Cl2TSV5R2; (lane i) sensitive
mouse L cell LMTK–; (lane j) colchicine (0.50 µg/
ml)-resistant mouse L cell ECHR; (lane k) sensi-
tive parent human lymphoid cell CCRF-CEM;
(lane l) vinblastine-resistant human lymphoid
cell CEM/VLB100 (see Table 1 for further de-
tails). The P-glycoprotein region is indicated (P)
at molecular size 150,000 to 170,000 daltons.
Lanes a to f and g to I represent two separate
gels that were run simultaneously and treated
identically. (B) SDS-PAGE was performed as in
(A), with the exception that 50 µg of membrane
protein was loaded per lane. Western blots were
overlaid with previously absorbed rabbit anti-
serum (diluted 1:100) against isolated plasma
membranes from the colchicine-resistant CHO
cells CHRC5 and processed as described (21,
27). Lanes a to f and g to l are two separate blots
prepared and treated identically, but lanes k and
l were exposed to film four times longer than
other lanes. All lanes are as described in (A). (C)
SDS-PAGE was performed as in (B), but blots
were overlaid with unabsorbed serum prepared
against CEM/VLB100 plasma membranes (di-
luted 1:100). Lanes k and I were exposed to film
for one-tenth the exposure time of other lanes.
drug resistant lines expressed a 170,000-dal- with different preparations of membranes. CEM/VLB100 (Fig. 1C). In this case, the serum
ton component that was stained by this an- The relatively faint staining of the presump- was not previously absorbed with membrane
tiserum (Fig. 1B). It is clear from the size and tive human P-glycoprotein by the antiserum proteins from drug-sensitive cells, and many
cross-reactivity of this component that it is is likely due to reduced cross-reactivity of this antigens common to both drug-resistant and
similar to the P-glycoprotein of the CHR CHO component with the antiserum against the drug-sensitive human cells were strongly
cells. Staining of other components of about CHO cell P-glycoprotein. stained (lanes k and l; note the exposure
50,000 and 200,000 daltons is also observed To further corroborate the above conclu- time). This crude antiserum stained only the
with this antiserum. We believe that these sions, we stained the membrane components drug resistance-associated P-glycoprotein in
components are not related to the P-glycopro- with an antiserum to plasma membrane of the rodent cell membranes (Fig. 1C). Thus, of
tein or multidrug resistance because they vary the vinblastine-resistant, human cell line, the dozen or so different cell surface antigens
that can be detected with the two antiserums, tance and surface P-glycoprotein in different Biophys. Res. Commun. 105, 157 (1982).
only the P-glycoprotein is consistently stained species of drug-resistant cells established in 19. W. T. Beck and M. C. Cirtain, Cancer Res. 42,
in all drug-resistant mammalian cell lines vitro. It is reasonable to suppose that such 184 (1982).
tested. These observations, and the fact that a relationship might also exist in vivo. If P- 20. R. L. Juliano and V. Ling, Biochim. Biophys.
a side-by-side comparison of the different P- glycoprotein is commonly present in tumor Acta 455, 152 (1976).
glycoprotein bands reveals no significant dif- cells from patient biopsies, and if this antigen 21. P. G. Debenham, N. Kartner, L. Siminovitch, J.
ference in molecular size, as shown in Fig. 1, is expressed in increased amounts in tumors R. Riordan, V. Ling, Mol. Cell. Biol. 2, 881
strongly indicate that the P-glycoprotein is nonresponsive to treatment by combination (1982).
conserved relative to other mammalian mem- chemotherapy, immunochemical screening 22. J. R. Riordan and V. Ling, J. Biol. Chem. 254,
brane antigens that are detectable by Western for the antigen could provide a rapid diag- 12701 (1979).
blotting. nostic basis for planning treatment of cancer 23. R. H. F. Peterson and J. L. Biedler, J. Supramol.
Our observations that P-glycoprotein is patients. Moreover, hitherto unresponsive Struct. 9, 289 (1978).
present in increased amounts in various drug- tumors may become amenable to treatment 24. V. Ling, Can. J. Genet. Cytol. 17, 503 (1975).
resistant lines, and that in the CHR CHO sys- with P-glycoprotein–targeted antibodies con- 25. __ and R. M. Baker, Somat. Cell Genet. 4,
tem the amount of P-glycoprotein expressed is jugated with toxins. 193 (1978).
correlated with the degree of resistance, are 26. R. M. Baker and V. Ling, in Methods in
consistent with a mechanism of resistance in- REFERENCES AND NOTES Membrane Biology., E. D. Korn, Ed. (Plenum,
volving gene amplification. The appearance of 1. N. T. Bech-Hansen, J. E. Till, V. Ling, J. Cell. New York, 1978), vol. 9, p. 337.
double minute chromosomes has been corre- Physiol. 88, 23 (1976). 27. N. Kartner, M. Shales, J. R. Riordan, V. Ling,
lated with unstable drug resistance in known 2. S. Brabbs and J. R. Warr, Genet. Res. 34, 269 Cancer Res., in press.
gene-amplified systems (28, 29). Double min- (1979). 28. P. C. Brown, S. M. Beverley, R. T. Schimke,
ute chromosomes have also been observed 3. L. J. Wilkoff and E. A. Dulmadge, J. Natl. Mol. Cell. Biol. 1, 1077 (1981).
in association with multidrug resistance in Cancer Inst. 61, 1521 (1978). 29. R. J. Kaufman, P. C. Brown, R. T. Schimke,
several mouse cell lines including colchicine- 4. B. Salles, J.-Y. Charcosset, A. Jacquemin-Sab- ibid., p. 1084.
resistant lines for which the degree of resis- Ion, Cancer Treat. Rep. 66, 327 (1982). 30. S. M. Robertson, V. Ling, C. P. Stanners, in
tance and P-glycoprotein expression correlate 5. T. Skovsgaard, Cancer Res. 38, 4722 (1978). preparation.
with the number of double minutes contained 6. V. Ling and L. H. Thompson, J. Cell. Physiol. 31. B. P. Kopnin, Cytogenet. Cell Genet. 30, 11
within the cells (14, 30). Double minutes have 83, 103 (1974). (1981).
also been reported in multidrug-resistant 7. K. DanØ, Acta Pathol. Microbiol. Scand. Suppl. 32. We have recently observed, using the
hamster cells that were originally selected for 256, 11 (1978). methods described, highly elevated
colchicine resistance (30, 31). This speculation 8. M. P. Chitnis and R. K. Johnson, J. Natl. P-glycoprotein expression in vincristine-
on the origin of the P-glycoprotein in drug-re- Cancer Inst. 60, 1049 (1978). resistant Chinese hamster cells, which bear a
sistant cells suggests its preexistence, in much 9. J. L. Biedler and R. H. F. Peterson, in Molecular chromosomal homogeneous staining region
smaller amounts, in the drug-sensitive parent Action and Targets for Cancer Chemothera- [see T. Kuo et al. in Gene Amplification, R. T.
cell. In this context, we observed a barely de- peutic Agents, A. C. Sartorelli et al., Eds. Schimke, Ed. (Cold Spring Harbor Laboratory,
tectable, antigenically cross-reactive band of (Academic Press, New York, 1981), p. 453. Cold Spring Harbor, N.Y., 1982), pp. 53-57].
the same molecular size as the P-glycoprotein 10. Y. Langelier, R. Simard, C. Brailovsky, 33. N. T. Bech-Hansen et al., J. Natl. Cancer Inst.
in all the drug-sensitive cells examined in Differentiation 2, 261 (1974). 59, 21 (1977).
Fig. lB when the film was exposed for a much 11. P. D. Minor and D. H. Roscoe, J. Cell Sci. 17, 34. R. C. Switzer, C. R. Merril, S. Shifrin, Anal.
longer period (data not shown). What spe- 381 (1975). Biochem. 98, 231 (1979).
cific role the P-glycoprotein might play in the 12. V. Crichley, D. Mager, A. Bernstein, J. Cell.
maintenance of the multidrug resistance phe- Physiol. 102, 63 (1980). ACKNOWLEDGMENTS
notype, or in the drug-sensitive cells, is un- 13. C. D. Aldrich, J. Natl. Cancer Inst. 63, 751 Supported by grants from the National Cancer
known. It is also not yet known whether the (1979). Institute of Canada and the Medical Research
P-glycoprotein is expressed in normal tissues. 14. F. Baskin, R. N. Rosenberg, V. Dev. Proc. Council of Canada. We thank W. T. Beck, P. G.
Our findings could have important implica- Natl. Acad. Sci. U.S.A. 78, 3654 (1981). Debenham, T. Kuo, and R. Simard for making
tions for cancer therapy. It is possible that clin- 15. S. A. Carlsen, J. E. Till, V. Ling, Biochim. available their drug-resistant cell lines; and N.
ical resistance to combination chemotherapy Biophys. Acta 455, 900 (1976). Alon, S. Fahim, and M. Naik for technical
might result from an unchecked proliferation 16. M. E. Lalande, V. Ling, R. G. Miller, Proc. assistance.
of tumor cell subpopulations with a multi- Natl. Acad. Sci. U.S.A. 78, 363 (1981).
drug resistance phenotype (33). The present 17. D. Kessel and H. B. Bosmann, Cancer Res. 30, 13 May 1983
data indicate that there is a strong correlation 2695 (1970).
between the expression of multidrug resis- 18. D. Garman and M. S. Center, Biochem.
GE Healthcare
GE Healthcare
High- and sup
Reproducibility using the DeltaV

Amersham™ WB system DeltaVision E
Åsa Hagner McWhirter, Anita Larsson, Elisabeth Wallby, Anna Edman-Örlefors, Ylva Laurin and Ola Rönn high-resoluti
DeltaVision Elite is a
imaging system sp
Introduction for imaging challen
Amersham WB system is a fully integrated system for separation, rabbit anti-ERK1/2, diluted 1:5000) and anti-mouse and anti-rabbit
transfer, detection, and quantitative analysis of proteins that gives secondary Amersham WB antibodies labeled with Cy3 (diluted • Improved contr
consistent, quantitative data for every sample, every time. The system 1:2500). The gels and membranes were scanned with optimal • More efficient li
achieves this high quality of data by combining three elements: settings in the system and the images evaluated using Amershamexposure condi
a SMART system design that standardizes and monitors every step WB evaluation software. The data was exported to Microsoft® Excel®
of the process; dedicated consumables and optimized protocols • graphs.
for coefficient of variation (CV%) calculations and linearity Fully integrated
that minimize assay variability; and built-in data normalization. The Un-labeled CHO cell lysate or HeLa and NIH 3T3 cell lysates (Santa platfor
system delivers consistent, quantitative data with fewer technical • Modular
Cruz) dilution series were used to compare reproducibility of target
replicates, very little hands-on intervention and less overall time expand capabil
signals. For chemiluminscence detection, Amersham ECL Gel 12%
required to generate results. and ECL™ Box electrophoresis unit (both from GE Healthcare) were flexib
Maximum
Whereas traditional manual Western blotting can take up to 24 h/run used for the SDS-PAGE. The proteins were transferred using wet transfer
or experiment, the Amersham WB system delivers Western blotting (TE22, GE Healthcare) onto Amersham Hybond™ P PVDFDeltaVision
membrane Elite ca
results, based on proven methods, and automatic evaluation of (GE Healthcare). ERK1/2 was targeted using primary rabbit including
anti- widefield
membrane image in just 4 to 5 h. The system uses fluorescence ERK1/2 (Sigma) diluted 1:20 000 (incubated overnight at multipoint
+4°C) and cell trac
detection to enable a wide dynamic range, excellent sensitivity, secondary HRP conjugated anti-rabbit IgG (GE Healthcare) diluted
(TIRF), fluorescence
and color multiplexing for reliable normalization and applications 1:100 000 and finally the signals were detected using Amersham
photokinetics, and
such as the simultaneous detection of phosphorylated/non- ECL Select detection reagent (GE Healthcare) and a CCD camera
phosphorylated proteins. The primary antibodies that work with (ImageQuant™ LAS 500 Imager, GE Healthcare). The signal Deconvolution
intensity s
traditional Western blotting protocols will work on the Amersham for each sample amount was determined using ImageQuantTL
Deconvolution imp
WB system, preserving a link to valuable historical data. The image analysis software (GE Healthcare).
Amersham WB system enables researchers, staff, and students contrast without sa
For fluorescence detection, the Amersham WB system with default
new to Western blotting to generate high-quality, quantitative exclusive deconvol
settings was used as described above. Tubulin was targeted using
data from day one, since the ease of use is high and there is error in discovery resear
primary rabbit anti-Tubulin (Sigma) diluted 1:1000 and secondary
proofing in a majority of the steps. Amersham WB Cy3 labeled anti-rabbit IgG (GE Healthcare) part of the system.
diluted
Here we present results for SDS PAGE and Western blotting 1:2500. The signal intensity for each sample amount was enabling
determinedstudies at
showing high reproducibility within or between gels/membranes using Amersham WB evaluation software (GE Healthcare). For more
for different users, both experienced and first time users. details, see figure legends. Outstanding foc
imaging experim
Methods UltimateFocus auto
Protein mixes or Chinese hamster ovary (CHO) cell lysates (prepared
Conclusion regardless of mech
at GE Healthcare) were pre-labeled with Amersham WB Cy™5 impact your experi
according to SDS PAGE and Western pre-labeling protocols. Amersham WB system is very reproducible and user
of UltimateFocus d
independent with built-in total protein normalization which
The samples were applied to Amersham WB gel cards. The
objective
can efficiently correct for uneven sample loading. This results and the c
electrophoresis, transfer to Amersham WB PVDF cards and probing
in CVs between 5 and 10% compared with a traditional objective into the a
was performed using the default settings of the Amersham WB system.
Tubulin and ERK 1/2 was detected using primary antibodies from manual workflow where CVs can be between 15 andor a camera.
35%.
Sigma-Aldrich (mouse anti-tubulin, diluted 1:1000 or 1:2500 and
Technical note
31
Linearity of Cy5 total protein pre-labeling Reproducible Cy5 pre-labeling of proteins Traditional manual workflow results in high
variation of target signals
MW
Cy5 total protein signal (volume × 106) 120 Markers
100
225 kDa (A) Concurrent (B) Non-concurrent
ECL detection ECL detection
97 kDa
80 R2 = 0.98833 Conalbumin CV: 4.3 % 400
66 kDa 500
350 16% 27%
60 50 kDa
Signal intensity (× 105)

Aldolas CV: 7 % 300 400
35 kDa
40 250
25 kDa 300
200 34%
20 20 kDa Ratio 22%
Conalbumin/Aldolase 150 200
14 kDa CV: 5.7 %
0 100 35%
10 kDa 16% 100
0 5 10 15 20 25 µg 50 35%
20%
0 0
Fig 1. Samples containing 10, 20, 30 or 40 µg of Fig 2. 14 Individual samples containing 2 µl 2.5 1.3 0.6 0.3 2.5 1.3 0.6 0.3
CHO cell lysate in 80 µl Mammalian Protein Extraction with a mix of Conalbumin and Aldolase NIH/3T3 cell lysate (µg) NIH/3T3 cell lysate (µg)
Buffer (GE Healthcare) containing protease inhibitor (0.1 µg/µl) were pre-labeled with Cy5 in 20 µl Fig 4. A dilution series of NIH/3T3 cell lysate was
mix (GE Healthcare) were pre-labeled for 30 min Amersham WB labeling buffer for 30 minutes applied a SDS-PAGE gel (Amersham ECL gel/
in room temperature. The reactions were stopped at room temperature. The reactions were ECL gel box, GE Healthcare) and the proteins were
by adding 80 µl sample loading buffer followed by stopped by adding 20 µl sample loading buffer, transferred to a PVDF membrane. The membranes
boiling at 95°C for 5 min. Samples containing 5, heated to 95°C for 5 min, and applied to an were blocked, probed and washed manually using
10, 15 or 20 µg of CHO cell lysate were applied in Amersham WB gel card, 8-18%. CV% of the trays and a rocker. ERK1/2 was targeted using
triplicate to an Amersham WB gel card, 8-18%. The signal intensity for each reaction as well as primary and secondary HRP conjugated anti-rabbit
Cy5 total signal within each lane on the membrane the ratio between the two protein signals IgG and the signals were detected using ECL Select
was plotted against the protein amount loaded. were calculated. detection reagent and a CCD camera. The signal
intensity for each sample amount was determined
and the average and CV% was calculated using
Microsoft Excel.
Comparing variation of non-normalized and normalized target signals
Six membranes were analysed concurrently (A) or
Cy3/Cy5 Cy3 Non-normalized Cy3/Cy5 on three separate occasions (B) but otherwise using
Image overlay Target signal (CV%) Normalized ratio (CV%) identical conditions. Sample dilution series, antibodies,
probing protocol, ECL reagent and detection exposure
15 000 8.0 0.20 5.4 time were identical for the signals to be compared.
0.15
User 1
10 000
0.10
5000 Amersham WB standardized, fluorescence based

0.05 workflow results in low variation of target signals
0 0
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 (A) Concurrent AWB (B) Non-concurrent AWB
Fluorescence detection Fluorescence detection
200 3500
15 000 6.7 0.12 5.0 5.9% 7.1%
175 3000
0.10

150
2500 7.5%
0.08 125 5.4%
10 000
User 2
2000
100 7.5% 7.2%
0.06 1500
75
6.4%
1000
5000 0.04 50 6.6%
25 500
0.02 6.2%
0 0
0 0 5.0 2.0 1.0 0.5 0.1 10.0 5.0 2.5 0.6
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 HeLa cell lysate (µg) CHO cell lysate (µg)
Fig 5. A dilution series of HeLa cell lysate was applied

30 000 0.30 to an Amersham WB gel card, 8-18%, and the
11.2 6.6
proteins were transferred to a Amersham WB PVDF
0.25
card. The membranes were blocked, probed and
20 000 0.20 washed using standardized automated protocol
User 3
0.15 in the Amerhsam WB system. Tubulin was targeted

using primary antibody and secondary Amersham
10 000 0.10
WB Cy3 labeled anti-rabbit IgG. The signal intensity
0.05 for each sample amount was determined using
0 0 Amersham WB evaluation software and the average
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
and CV% was calculated using Microsoft Excel.
Fig 3. Three different first time users of the Amersham WB system performed Cy5 pre-labeling of CHO cell Four sample replicates on two membranes were
lysate and 7.5 µg total protein per well were loaded to an Amersham WB gel card, 13.5%. Western blotting analysed concurrently in the same Amersham WB
was performed targeting ERK1/2 with primary antibody and Amerham WB Cy3 labeled secondary run (A) or on three separate occasions (B) but otherwise
antibody. The target signals were normalized using the Cy5 total protein signal. The CVs were all reduced using same conditions. Sample dilution series,
compared to non-normalized target signals and below 7% for the normalized ratio (Cy3 target/Cy5 total antibodies, and the probing protocol were identical for
protein) indicating correction of the uneven loading. the signals to be compared.
www.gelifesciences.com
GE and GE monogram are trademarks of General Electric Company. Amersham, ECL, Cy, CyDye, Hybond and ImageQuant are trademarks of General Electric Company or one of its subsidiaries. Excel is a registered trademark of Microsoft
Corporation. All other third-party trademarks are the property of their respective owners.
CyDye: This product is manufactured under an exclusive license from Carnegie Mellon University and is covered by US patent numbers 5,569,587 and 5,627,027.
The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes. A license to use the CyDye products for commercial purposes is subject to a
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29133719AA 05/2015
32
What if Western blotting was
consistent and quantitative?
Introducing the fully integrated Amersham™ WB system designed
to deliver consistent quantitative data every sample, every time.
With reduced repetition and fewer control experiments, you will feel
confident that you have the results to move forward.
Go to www.gelifesciences.com/artofwesternblotting to find out more.
Amersham | Biacore | ÄKTA | Whatman | Cytell | Xuri*
GE and GE monogram are trademarks of General Electric Company.
* Amersham, Biacore, ÄKTA, Whatman, Cytell and Xuri are trademarks of General
Electric Company or one of its subsidiaries.
© 2015 General Electric Company—All rights reserved. First published May 2015.
GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
Amersham™ Western blotting:
expertise for results without
compromise
The Amersham Western blotting portfolio brings together Rainbow™ markers,
Hybond™ membranes, blockers, CyDye™ conjugated secondary antibodies,
Amersham ECL™ detection reagents, imaging systems and Amersham WB system.
Whether you need to confirm the presence of your protein or quantify multiple
targets with fluorescence detection, Amersham Western blotting has a
solution for your needs.
Go to www.gelifesciences.com/amershamwb to find out more.
Amersham | Biacore | ÄKTA | Whatman | Cytell | Xuri*
GE and GE monogram are trademarks of General Electric Company.
* Amersham, Biacore, ÄKTA, Whatman, CyDye, Cytell, ECL, Hybond, Rainbow
and Xuri aretrademarks of General Electric Company or one of its subsidiaries.
© 2015 General Electric Company—All rights reserved. First published May 2015.
GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK

Western Blotting: A Guide To Current Methods

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Western Blotting: A Guide To Current Methods

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A Sponsored Supplement to Science

Western blotting overview

7 Preparing for your Western blot

10 Choosing your detection method

22 A neuronal antigen in the brains of Alzheimer patients

25 Serological analysis of a subgroup of human

BILL MORAN, GLOBAL DIRECTOR

© 2015 by The American Association for the Advancement

Tianna Hicklin, Ph.D.

reproducible the ultimate goal and reproducible quantitative data is an absolute

Quantitative Western blotting:

Western blot tips and tricks:

A history of the old

West(ern blot) antibodies to a specific protein or proteins of interest, and de-

Ling’s team didn’t To find out, Robert

Preparing for your

The latter tends (TBST or PBST) (6),

Westerns, of course, retain protein conformation). You may need Good_Westerns.pdf].

Quantitative vs. qualitative detection

stripping can also remove and colleagues described

An analysis of critical factors for equal volume of dithiothreitol-containing

12 Originally published 7 April 2015 in SCIENCE SIGNALING sciencemag.org SCIENCE

Fig. 2. Posttranslational modifications can move protein into the

ever, a single loading control may not fulfill Fig. 3. Phospha-

Fig. 4. Linearity and hyperbolic saturation

removed by the recommended stain removal

high-affinity antibodies that work reliably

make extrapolations that could persistent concern is saturation

peptin (10 μg/ml), pepstatin (1 μg/ml), 1 mM immunoblots here, some phosphorylation

A neuronal antigen in the brains of

22 Originally published 2 May 1986 in SCIENCE sciencemag.org SCIENCE

were thioflavine-positive were in native form on a Sepharose

Fig. 4. Western blot of (A) Alzheimer and REFERENCES AND NOTES

human T-lymphotropic retroviruses

(HTLV-III) associated with AIDS antibodies to HTLV-III in AIDS and pre-

SCIENCE sciencemag.org Originally published 4 May 1984 in SCIENCE 25

Fig. 3. Relation between HTLV-

with multidrug resistance in

mammalian cell lines in each case a multidrug resistance pheno-

28 Originally published 23 September 1983 in SCIENCE

Fig. 1. Electrophoretic and immunochemical

High- and sup

Reproducibility using the DeltaV

Signal intensity (× 105)

Signal intensity (× 105)

5000 Amersham WB standardized, fluorescence based

Signal intensity (× 103)

Fig 5. A dilution series of HeLa cell lysate was applied

0.15 in the Amerhsam WB system. Tubulin was targeted

Amersham | Biacore | ÄKTA | Whatman | Cytell | Xuri*

Amersham | Biacore | ÄKTA | Whatman | Cytell | Xuri*

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