What is a Western Blot?

A method to detect protein in a complex mixture

of biomolecules using specific probes
(antibodies) having an affinity for a protein
Pre-immunobloting
Isolated protein, Protein assay,
Tracking dye
Electrophoresis-separation of proteins
Commasie staining
Electrophoretic transfer of proteins from
the gel to the membrane.
Ponseau S. dye




Ponseau S.

To check if equal amount were loaded
If similar proteins were visible
If the transfer procedure worked
Works under acidic conditions
Comes off under neutral conditions
Membrane Transfer
To make proteins accessible to antibody,
they are transferred onto a support where ag-
ab complex can form. Types of Membranes

Nitrocellulose
Fragile, cant stand repeated probing.

Polyvinylidene difluoride (PVDF)
Resistant to solvents therefore can be stripped and reprobed.

Both show non-specific affinity for proteins but how
proteins bind is not understood (combination of
electrostatic, hydrophobic and hydrogen bonds)

Membrane Transfer

Transfer sandwich from
cathode (-) to anode (+)
Sponge (s)
3 sheets filter paper soaked
in transfer buffer
Gel
Membrane
3 sheets filter paper soaked
in transfer buffer
Sponge (s)

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Membrane Transfer
Efficiency of Transfer
Protein binding capacity of membrane
Nitrocellulose (80 - 100 g/cm
2
)
PVDF (100 - 300 g/cm
2
)
Transfer method
Tank method
Semi dry
Buffer strength, pH, time, voltage
Nature of protein
Size (MW)
Hydrophobicity

Membrane Transfer
Efficiency of Transfer

SDS can interfere with binding, especially on PVDF membranes
by coating the protein molecule, the SDS limits the ability of the
protein to make molecular contact with the PVDF

SDS imposed problems increase as the MW of the protein decreases.

Methanol is added to strip off SDS and stabilize the dimensions of the
gel.

Pre-soak gel 5-15 min. in transfer buffer without SDS prior to transfer
to reduce SDS concentration and enhance binding
Membrane Transfer
Efficiency of Transfer

Hydrophobic proteins
Additional methanol
(up to 20%) in transfer
buffer



Bigger proteins
Run in a lower % gel
Increase transfer time
Additional 0.05% SDS
in transfer buffer
Lower or omit
methanol


Blocking Non-Specific Binding
Block remaining hydrophobic binding sites on membrane

Prevent binding of primary antibody to the membrane
itself

Block with Tris-HCl, 0.2%
tween and 4% non-fat milk
for 20 mins




Blocking Non-Specific Binding
Blocking solutions
3-10% w/v BSA
4-5% non-fat dried
milk
Incubate room temp.
or 4C overnight
Blocking solutions
should not contain
antigens that may be
recognized by the 1
or 2 antibody

Too much blocker can
mask the ag-ab
interactions or inhibit
the marker enzyme
Antibody Probe
Antibodies have been found to be the method of
choice in protein detection
Independent of biological activity
Sensitive enough to detect small amounts of protein
Primary ab is specific for actin

Antibody Probe
Primary Antibody
Recognizes the
protein of interest
Secondary Antibody
Binds to primary antibody
at the species specific
conserved region
Usually produced by a
different animal species
Antibody-enzyme
conjugate
Enzyme has a substrate
molecule that is converted
to a colored reaction
product
Can also carry radioactive
or chemiluminescence tag
Antibody Probe
Dilute the 1 antibody (serum) in Tris with or
w/o blocking protein (BSA)
Incubate with blot for 30 minutes (various
times/temperatures), with gentle agitation
Wash blot several times to remove unbound 1
antibody, using Tris/0.1% Tween
25ml 5mins
30 ml 5mins
30 ml 10mins
Repeat steps 1-3 with 2 antibody


Detection

Alkaline phosphatase or horseradish peroxidase
Conversion of colorimetric substrate to a colored
precipitate
Chemiluminescent substrates
Emit light upon conversion by the enzyme
Can be captured on x-ray film
Radioactive labels
Biotinylated 2 antibody


ABC-AP (Avidin-Biotin Complex with Alkaline Phosphatase)
ABC-AP substrate - BCIP/NBT
BCIP (5-bromo-4-chloro-3-indolyl phosphate)
NBT -- nitroblue tetrazolium
BCIP acts as the substrate for alkaline phosphatase
Alkaline phosphatase removes Pi from BCIP
Oxidized BCIP dimerizes and precipitates
donates e
-
to Nitro-blue tetrazolium, which precipitates
NBT enhances the purplish-brown final color of the precipitates
at the reaction sites


Color Development
Wash 3X with wash buffer
30 ml 5min (2X)
30 ml 10 min (1X)

Wash 1X with 25 ml of 100ml Tris-Cl, pH 9.5
Submerge in color development soln
Look for purple bands
Stop color development by washing with water
Western blot stains for the presence of
antibody-antigen complexes
Original gel stained
for proteins
gel
nitrocellulose
Western blot stained
for antibody-antigen
Antibody Probe



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Troubleshooting
Streaking of blots =overloaded
protein
Lack of staining = ineffective
sample transfer from gel to blot


Poor transfer
Air bubbles between gel
and membrane
Membrane on wrong side
of gel in reference to
current
Insufficient current/time
SDS concentration too high
in transfer buffer
www.ruf.rice.edu
Troubleshooting
Weak Staining
Insufficient 1 antibody
incubation time
1 antibody too dilute
Improper storage of
reagents
Kept at RT
Repeated freeze/thaw
High Background
Incomplete blocking
Excess antibody
concentration
Insufficient washing
Expired/contaminated
reagents
Excess exposure time
Troubleshooting
Multiple bands
May indicate reaction
with isoforms of an
antigen that happens
to possess common
epitopes
Non-specific reactivity
with unrelated proteins

Unanticipated size
Multiple bands near
expected MW of
antigensign of
incomplete reduction
Lower MW sign of
proteolytic degradation