Western Blotting

Dr. Usman Ali Ashfaq

Dept. of Bioinformatics and Biotechnology, GC University,
Faislabad

Dept of Bioinformatics and Biotechnology, GC University, Faislabad

Western Blot

•The western blot is a widely

accepted analytical technique used to
detect specific proteins in the given sample
of tissue, homogenate or extract

• Method developed by George Stark

• The name Western blot was given to the

technique by W. Neal Burnett

Dept of Bioinformatics and Biotechnology, GC University, Faislabad

 Western blotting
• Detects proteins and estimates their
molecular weight.
• Detects changes in phosphorylation and
lipid modifications.
• Used to detect changes in protein
expression.
Protein Lysis Buffer

• Buffer (Tris or Hepes buffer with pH 7-8)

• Salt (usually NaCl 150mM (low) to
500mM(high)
• Chelating agent (EDTA or EGTA)
• Detergent
• Protease inhibitor
• Phosphatase inhibitor (optional)

Dept of Bioinformatics and Biotechnology, GC University, Faislabad

 Lysing Cells
• Treat cells with appropriate conditions depending on
the experiment
• Pellet cells and lyse them in the appropriate lysis
buffer.
• Most important ingredient in lysis buffer is detergent.
– Most stringent to weakest
• SDS
• NP-40
• Triton X-100
• Tween 20
• Digitonin
• CHAPS
• Brig
 SDS covers proteins in a net negative charge
Addition of 2-mercaptoethanol reduces disulphide bonds and
Boiling is used to further denature proteins.

Charged R groups
+
-
-
-
+ +
H
-

+
+ Hydrophobic areas
H
+
-
-

-
- -
Before SDS
- - -
-
- - - -
- - - -

Migrate in gel according to mass

• Second most important ingredient is protease
inhibitors.
– Once proteins are denatured by detergents, they are
susceptible to degradation by proteases.
– Need more than one inhibitor since there are lots of
proteases.
– Protease inhibitors
• Aprotinin
• Leupeptin
• PMSF (phenylmethylsulfonyl fluoride)
– Add immediately before lysis since PMSF activity decreases
over time in aqueous solutions. About 15-30 minutes of activity.
 Phosphatase inhibitors
• Need to add to lysis buffer if using
phopshospecific antibodies or suspect protein of
interest is phosphorylated
• Inhibitors
– ZnCl2
– NaF
– Na-Vanadate (tyrosine phosphatase inhibitor, add
prior to lysis since only active in pH 7 for minutes)
 Further denature proteins
• Add lysate either a known concentration of proteins or
cell number equivalent to SDS loading buffer
• SDS Loading Buffer
– Buffer (Tris-Cl pH 6.0)
– 2% SDS
– 0.1% bromophenol blue
– 10% glycerol (allows sol’n to sink to bottom of gel wells)
 -mercaptoethanol ( reducing agent)
• SDS loading gel mixed with lysate is boiled to further
denature proteins.
• 1:10 ratio loading buffer to lysate
 SDS-Polyacrylamide Gel
Electrophoresis of proteins
• This gel ensures that proteins are
dissociated into their polypeptide subunits
and minimizes aggregation.
• Proteins migrate on gel according to their
molecular weight therefore if proteins of
know amounts are added to the gel the
approximate molecular weight of your
protein of interest can be found.
 SDS-Polyacrylamide Gel
Electrophoresis of proteins
• Protein lysate is now loaded onto a SDS-
polyacrylamide gel electrophoresis apparatus.
• This apparatus contains a gel that separates
protein out according to their molecular weight.
• GEL
– 15% 12-43kDa
– 10% 16-68kDa
– 7.5% 36-94kDa
– 5.0% 57-212kDa
 Ingredients in Gel
• Sodium dodecyl sulfate (SDS)
• Tris buffer (either glycine or tricine)
• Acrylamide and NN-bis-acrylamide
– Forms gel matrix
• TEMED
– Catalyst for polymerization (produces free radials from APS)
• Ammonium persulfate (APS)
– Source of free radials for polymerization

Could purchase pre-cast gels if you have the money.

Transfer of proteins to the membrane
Western blot-semi-dry transfer of proteins
 After transfer
• Membrane is washed in a Tris, borate, saline
solution.
– Options
• Added detergent Tween 20 to reduced background in western
blotting.
• Block membrane in non-fat dried milk solution.
Usually 5% w/v. Prevents binding of antibodies to
membrane.
– Don’t buy milk from Fisher
– Safeway brand or Carnation brand are the best
– Alternatively, use bovine serum albium (BSA) but its
more expensive (not available in Supermarkets)
 Antibodies
• Proteins that bind to specific epitopes on specific
proteins.
• Two main types used in western blotting
– Monoclonal
– Polyclonal
• Monoclonal are generally produced from mice
and polyclonal are produced from rabbit or goat
but other animals also produced polyclonals.
 What antibodies are best for
western blotting?
• Both types work for western blotting but
generally polyclonals are better
– Monoclonals are to one epitope while
polyclonals are to many epitopes on the
protein of interest. If epitope changes upon
denaturing of the protein it will not recognize
the protein
– Each antibody has its own characteristics so it
is hard to predict what antibodies work well in
a western and which are bad.
 Primary Antibodies
• After blocking membrane, add antibodies in
concentrations recommended by manufacture or
in dilution series if unknown to blocking solution
(ie 5% milk).
• Most antibodies are added as a dilution such as
1:1000.
• Incubate overnight at 4oC or 2 hours at room
temperature (only if it’s a good antibody).
• Wash in TBS with detergent Tween 20 (TBST)
 Secondary Antibodies
• Wash at least three times with TBST for 5-10
minutes each
• Added antibodies against the animal that the
primary antibodies were made in.
• These antibodies are also conjugated with
enzymes such as horse radish peroxidase
• The antibodies are added to blocking buffer and
incubated at room temperature for one hour.
 Detection of your protein
• After secondary antibody incubation wash
with TBST as before.
• Add enhance chemiluminance reagents
(commercially available) that have
substrates for HRP that gives a product
that gives off light energy.
• Place on autorad film and bands appear.
Detection
Primary antibody followed by:
Radioactive-labelled
125
I staphlococcal protein A or streptococcal protein G
Enzyme-linked secondary antibodies
-horseradish peroxidase (HRP)
-alkaline phosphatase-BCIP/NBT
BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) and
NBT (Nitro-Blue Tetrazolium Chloride).

Chemiluminescent detection-
HRP catalyzes the oxidation of luminol in hydrogen peroxide.
Luminol decays by light emission.

AP catalyzes the dephosphyorylation of adamantyl-1-2-dioxetane

phosphate, resulting in emission of light.
Southwestern blot
Figure 7 Distribution of the 52 kDa protein in various mouse tissues as
analysed by Southwestern blot analysis

Biochemical Journal (1998) 329, 623-629 -

www.biochemj.org