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The Popular Technology SDS

PAGE & Western


blotting :Principle
and Application

Current Protocols in Molecular Biology 10.8.1-10.8.28, July 2008

Western Blot Definition:

Western blotting is a technique used to identify


and locate proteins based on their ability to bind to
specific antibodies.

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Western blot analysis can detect your protein of
interest from a mixture of a great number of
proteins (a cell can contain 30,000 different
proteins - and these same proteins can even be
altered giving you over 300,000 different proteins!).

an idiom in Chinese

(大海撈針)
Western blotting can give you information about
the size of your protein (with comparison to a size
marker or ladder in kDa), and also give you
information on protein expression (with comparison
to a control such as untreated sample or another cell
type or tissue).

Summary: Western Blot Gives You Information on the:


· Size of your Protein
· Expression Amount of your Protein

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Western blot analysis can analyze any protein sample
whether from cells or tissues, but also can analyze
recombinant proteins synthesized in vitro.

Western blot is dependent on the quality of antibody


you use to probe for your protein of interest, and how
specific it is for this protein. Antibodies are now easily
obtainable from commercial sources, and you can
purchase one for your protein of interest.

Antibodies specific to your protein are vital to western


blotting as they are able to bind specifically to your
protein of interest instead of the thousands of proteins
on your western blot!

Figure 1. Details of the steps involved in obtaining protein for western blot.

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Definition: SDS-PAGE is an abbreviation for sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)
Electrophoresis is a technique for separating,

SDS-PAGE
• Standsfor “SDS Polyacrylamide Gel Electrophoresis
• Similar in many ways to agarose gel electrophoresis
that we used to separate DNA molecules:

1)Thin gel separates molecules on the basis of size.

2) Negatively charged molecules are loaded into wells


in a gel that is submerged in buffer.

3) Electrical current is applied to the gel, causing the


molecules to move toward the positive electrode (the
anode, usually marked red)

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SDS-PAGE
4) Smaller molecules move faster through the gel than
larger molecules

- Thus, smaller molecules will be found at the


bottom of the gel and larger molecules will be
found at the top of the gel.

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In forming the polyacrylamide gel, acrylamide monomers polymerize into long chains that
are covalently linked by the crosslinker N, N'-methylene-bis-acrylamide (bis for short).

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Analysis of protein samples by SDS polyacrylamide-
gel electrophoresis and Western blotting

Protein
bands
detected by
specific
antibody

SDS-PAGE Western blot

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To Detect your Protein:

· Buy an Antibody Against Your Primary Antibody

· Use an ECL - Chemiluminescence Kit and Film to


Get the Results

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Table of Contents, in order done for western blot:
Steps in Western Blotting:

-Preparing for Western Blot

-Which Antibody to use for Western Blots

-Lysing Cells for Western Blot

-SDS-PAGE Gel Information

-SDS-PAGE Gel Preparation for Western Blots

-Gel Electrophoresis - running the gel

-Tranfer of Proteins to Membrane for Western Blotting

- Western Blot

To Prepare Samples for Western Blot:

Whether you have Non-adherent Cells (such as T-cell


lines or peripheral blood cells) or Adherent cells (such
as Fibroblast cells or COS cells), you need to remove
extraneous proteins from the media.

For non-adherent cells you can gently pellet them


with centrifugation and then wash the pellet with PBS.
For adherent cells, simple wash the flask or dish with
PBS prior to western blot.
Western blot analysis examines proteins, and so we
want the proteins to be release from the cells and
also prevent the proteins from being cut up by
proteases.

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To Prepare Samples for Western Blot: continue

We need these to western blot:


- to keep things cold! 4C on ice during cell lysis for western
blotting
- use Detergent to break up membranes of cells to release
proteins
- Buffer
-Inhibitors (both protease inhibitors and phosphatase
inhibitors if we will do western blot for phospho-proteins)

Detergent - Lysing Cells For Western Blot


SDS and RIPA are detergents that are used to solubilize
proteins for later analysis using western blotting. This is
required as many proteins are either inside the cell or located
inside cell membranes, so we need to release these proteins
to be able to immunoblot for them.

Which Antibody should I use for Western Blotting?

You should select an Antibody to use for Western Blot. Usually


either mouse monoclonal antibodies or rabbit polyclonal
antibodies are used.
You can use either an antibody without any added groups, or use
an antibody which is conjugated to biotin.

Monoclonal Antibodies are usually better for Western Blotting due


to:
- better signal to noise ratio ( lower background in western blot )
- their higher specificity (you get less contaminating proteins or)
- overall cleaner results on the western blotting film (less non-
specific bands detected)

Polyclonal Antibodies are better suited for Immunoprecipitation:


- they recognize more epitopes
- have higher avidity (親合力)

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Lysing:

-can be done directly on the plate or dish

-pellet the cells

-Keep on Ice and cold - use an ice bucket

-Rule of thumb for how much lysis buffer to use is:


10^5 cells /uL of lysis buffer

- collect in eppendorf tubes and label (keep these


on ice )

Measure total protein before Western Blot


Analysis:

-this allows more quantitative analysis if you want to


compare treatment conditions in western blot
analysis

-commercial kits are available such as a Bradford


assay for measuring total protein (from Bio-Rad)

- 0.1% of SDS or greater can interfere with protein


measurement

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Denature Protein Samples :

-add protein loading sample buffer to an aliquot of


cell lysate - contains dye (too see the migration on a
gel, 2% SDS)
-add disulfide reducing agent such as beta –
mercaptoethanol

- Boil in water bath or heating block (lower


temperatures such as mid - 90 degrees decrease
protein aggregation ).
- Poke a hole in cap of each tube to prevent
popping

SDS-PAGE Gel Information for Western


Blotting
SDS-PAGE Gels are gel matrices which are used to
separate proteins by size in the presence of electric
current. Low percentage gels separate larger proteins
whereas higher percentage gels separate smaller
proteins better.
The problem is that all proteins have a charged
associated with them, and in an electrical current this
could cause problems. This is solved by the addition
of SDS to the protein samples. SDS binds to proteins
every few amino acids and neutralizes the charge
differences that proteins have. This allows proteins to
be separated by size and not by charge.

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SDS-PAGE Gel Preparation for Western Blot

- depending on how much protein you will want to load for


western blotting, you should use small combs or larger combs.
- the percentage of acrylamide is important. Usually 10%
acrylamide is used.
- A resolving gel is used at the bottom with a pH of 8.8
-A stacking gel (4-5%) pH 6.8 is used to pack proteins in
together after loading

- Polymerization of gels is increased by the catalysts APS and


TEMED which speed up the polymerization reaction (formation
and solidification) of the poly-acrylamide gel.
- Load every Sample carefully and slowly to prevent sample
leaking out of the lane.
- Load every lane and use sample buffer in each (to prevent
differences in ).

Percent acrylamide Size range transferred


(resolving gel) (∼100% efficiency)

5–7 % 29–150 kD
8–10 % 14–66 kD
13–15 % <36 kD
18–20 % <20 kD

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Gel Electrophoresis

Centrifuge samples for 10 sec after boiling.

Load sample into each lane

Load MW marker

Run gel at 100V (constant voltage) or even better at 40


mA (constant current).

Watch protein marker/ladder or dye front for when to


stop gel.
Constant current gives better and sharper results if you
have the time.

Gel Electrophoresis

-watch for bubbles between glass and under the gel -


this means that it is working

-watch for protein migration (should be going down) - if


not you have switched the leads and can lose all your
samples!

- Initially run slowly through the stacking gel (~ 50


Volts), this gives sharper bands.
- Do not run overnight
- Do not over-heat the gel (this causes the gel to lose
rigidity, leading to poor resoloving and blurry badly
formed bands)

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How an SDS-PAGE gel is run

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Tranfer of Proteins to Membrane for Western
Blotting

Select Membrane Type:


Can use either:

-PVDF membrane for western blots (hydrophobic)

-Nitrocellulose membrane for western blots


-
-Nylon membrane (rarely used now - can tear)

Tips for Transfering to Western Membrane:

-Wear Gloves at all times! Use forceps


- Minimize touching/forceps to the membrane

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Assemble the immunoblot sandwich

(-)
sponge on black
?
PVDF membrane
filter paper

Gel

filter paper

sponge

(+)

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How Does Western Blotting Work?

See Diagram 1 below.

Obtain a protein sample you want to analyze, such as cell samples.


Lyse the cells to release protein contents.
Run these on a gel which separates proteins on the basis of size.
Then transfer these gel proteins onto a membrane using electricity.
This membrane can then be used to probe for proteins of interest using a
primary antibody.

What You Need to Western Blot:

· A Protein Sample
· A Good Antibody to Detect your Protein of Interest If your protein is a novel
protein, you must produce an antibody yourself or get a company to do it for you. In this case you
will need at least a small amount of your protein either purified from cell extracts or made as a
recombinant (ie in vitro or in a recombinant protein expression system).

Primary antibody

Wash

Secondary antibody

Wash

detection

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Diagram 1

Membrane (carrier)

Diagram 2

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Diagram 2 shows a western blot example gel.

Lane 1 is a protein size marker ladder which shows different


known sizes of proteins, this can be purchased commercially and
the sizes of all the spots are given.

Lane 3 is a cancer sample and


lane 5 is a normal sample.

As you can see the protein in lane 3 has a higher expression than
the normal sample in lane 5, which is interesting.

Also, the protein spots in lanes 3 and 5 are the same size as the
2nd spot in the size ladder from lane 1.

Our protein of interest is also 80 kDa. So we know that the


western blot worked and that the protein is highly expressed in a
cancer sample!

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If the membrane is placed into the water too quickly, air
will be trapped and will appear as white blotches in the
membrane. Protein will not transfer onto these areas.
Place into distilled water slowly, with one edge at a 45◦◦
angle. This wetting procedure works for nitrocellulose and
nylon membranes only.

PVDF membranes are hydrophobic and will not wet


simply from being placed into distilled water or
transfer buffer. For these membranes, first immerse 1 to 2
sec in 100% methanol, then equilibrate 10 to 15 min with
transfer buffer. Do not let membrane dry out at any time.
If this occurs, wet once again with methanol and transfer
buffer as described above.

Assaying for Results


-usually ECL (Enhanced Chemiluminescence) is used

- film is exposed and developed. Exposure for 10 seconds


is usually enough for total protein. Phospho-proteins can
require 2 - 20 minute exposures.

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chromogenic

Chromogenic- substrate

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Chromogenic detection

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Table 10.8.3 Applications of Immunoblotting

Application Comment

Antibody development
and characterization Characterize antibody specificity

Subcellular
localization of an
expressed protein Analyze isolated organelles (e.g., Golgi apparatus, plasma
membrane, nucleus) for presence of specific proteins; probe
intact cells and tissue for subcellular localization.

Protein purification Demonstrate enrichment of well-characterized antibody by


sampling, electrophoresing, and blotting at various stages of
purification

Diagnostics Separate and blot a viral lysate to test serum for antibodies
that bind key viral proteins, indicating presence of antibodies
against an infection (e.g., HIV testing)

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Application Comment

Gene expression Detect presence or absence and amount of a specific protein


during gene expression; track markers and reporter genes (e.g.,
luciferase) in transgenic organisms to get a complete picture of
transcription and translation
Post-translational
Modifications Phosphoproteomics: determine phosphorylation status of the
protein complement in the cell or tissue using antibodies
specific for phospho amino acids

Protein sequencing by
mass spectrometry Characterize the readily available proteins blotted onto
membranes using sensitive methods, e.g., peptide sequencing
by MALDI (Carr and Annan, 1996)

Troubleshooting Western Blot

Spots on Film (missing bands) :

-Poor Transfer due to air bubbles during transfer to


membrane

-dirty film when adding to the developer

- developer rolls are dirty

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Fuzzy Bands, Bands Smeared, Bands not Sharp

-bands smeared due to hot gel

- bands fuzzy due to high voltage

STRIPPING AND REUSING MEMBRANES

Reprobing PVDF membranes that have been developed with


chemiluminescent reagents is simple and straightforward. All
residual antibodies are removed from the membrane by first
rewetting it in water and then briefly treating it with NaOH.
Although repeated reprobing can lead to loss of signal, up to
five reprobings are generally feasible. The blot should have
been blocked with 5% nonfat dry milk prior to treatment.

Chromogenic development leaves a permanent stain on the


membrane that is difficult to remove, and should not be used
when reprobing. The stain can interfere with subsequent
analysis if reactive bands from sequential immunostainings
are close together.

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Materials
0.2 M NaOH

1. Wash blot 5 min in distilled water.


In order to effectively reprobe the membranes, casein (for
AP systems) or nonfat dry milk must be used as the blocking
agent.

2. Transfer to 0.2 M NaOH and wash 5 min.

3. Wash blot 5 min in distilled water.

4. Proceed with immunoprobing procedure (see Basic


Protocol 2 and Alternate Protocol 4).

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Flow chart of Western blotting

Electrophoresing the protein sample

Assembling the Western blot sandwich

Transferring proteins from gel to


nitrocellulose paper

Staining of transferred proteins (skip or not)

Flow chart of Western blotting


Blocking nonspecific antibody sites on the PVDF paper

Probing electroblotted proteins with primary antibody

Washing away nonspecifically bound primary antibody

Detecting bound antibody by secondary antibody


conjugated with horseradish peroxidase and

Use an ECL - Chemiluminescence Kit

Developing the immunoblot

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