IMMUNOBLOTS

Types

• Western blot- protein analysis

• Northern blot- RNA analysis
• Southern blot- DNA analysis
 Western Blot

• Definition
• Principle
• Procedure
• Clinical utility
• Advantages
• Limitations
 Definition

• It is a molecular technique used to separate and identify

specific proteins from a mixture of proteins present in a
tissue or cell
– Also known as protein immunoblot
• It can also be used:
– to evaluate the size of a protein of interest and
– to measure the amount of protein expression
(quantification)
 Principle of WB
• Separation of proteins in a sample;
– achieved through gel electrophoresis following their denaturation
• Separated proteins are then transferred to a solid
support/nitrocellulose membrane (blot)
• Target proteins are identified by the use of protein-specific
labeled monoclonal antibodies (primary and secondary)
• The result is formation of protein bands that are visualized by
either:
– immunostaining,
– immunofluorescence or
– radioactivity
• Band thickness corresponds to amount of protein present and
when compared with a standard can be used for quantification
 Procedure
• Western blot involves the following steps:
1. Sample preparation
2. Gel electrophoresis
3. Transfer/Blot
4. Staining
5. Blocking
6. Incubation
7. Detection and visualization
 Sample preparation
• Proteins are extracted from cells using ice-cold cell lysis buffer
with a protease inhibitor cocktail (prevents catalysis of proteins)

– The protein sample thus obtained is denatured by heating at 100°C for

5 minutes and by use of a reducing agent e.g. mercaptoethanol
• The protein sample is then mixed with sodium dodecyl sulfate
(SDS)
– This makes the proteins unfold into linear chains and coats
them with a negative charge (they become anionic)
– NB – Include positive and negative controls (purified target protein; null
cell line/structural protein e.g. actin or tubulin)
 Gel electrophoresis
• Proteins are separated according to their sizes using SDS
buffer and polyachrylamide gel electrophoresis (PAGE)
• Protein samples mixed with a tracking dye (bromophenol blue)
are loaded into wells in the gel and a voltage is applied along
the gel
– The smaller the protein, the faster it migrates (negative to positive)
– Different rates of migration separate the proteins into bands within
each lane (runtime = 1 hour or until tracking dye reaches the end)
• One lane is reserved for a marker/ladder (proteins mixture of
known Molecular weight) for comparison with sample proteins
to estimate their MWs
 Transfer/Blot
• Separated proteins are transferred from the gel onto a membrane
made of either nitrocellulose (NC) or polyvinylidene difluoride
(PVDF)
– Proteins are transferred either by electroblotting or capillary action
• Capillary action involves use of sponges & filter papers to make a
sandwich with the gel and membrane;
– transfer buffer is used
• Electroblotting uses the same setup as capillary action but with an
electric current to pull the proteins from the gel onto the membrane
 Staining
• Total protein staining (TPS) allows the total protein that has
been successfully transferred to the membrane to be visualized

• In order to avoid noise of signal (background), TPS should

ideally be performed before blocking of the membrane

• The most common stain used for TPS is: Coomassie Brilliant
Blue.
 Blocking
• Treatment of the membrane to prevent antibodies from binding non-
specifically, instead of their target proteins

• Blocking is achieved by placing the membrane in a dilute solution of

protein – typically:
– 3–5% bovine serum albumin (BSA) or
– non-fat/skim dry milk in tris-buffered saline (TBS) – for 1 hour

• The protein in the dilute solution attaches to the membrane in all places
where target proteins have not attached

• Thus, when antibody is added, it cannot bind to the membrane, and

therefore the only available binding site is the specific target protein
 Incubation
• The membrane is then incubated with primary antibody, which
specifically binds to the target protein
– Primary antibody is incubated with the membrane under gentle agitation
for an hour at room temperature or overnight at 4°C
• Following incubation, any unbound primary antibody is washed
away, and the membrane is incubated yet again, but this time:
– with a secondary antibody that specifically binds to the primary
antibody.
• Unbound secondary antibody is also washed away
• The secondary antibody enhances or amplifies the signal
 Detection and visualization
• To detect the presence of the target protein, the secondary
antibody is linked/bound to a label or tag that produces either:
– color (colorimetric),

– light (chemiluminescence, fluorescence) or

– radioactivity, which allows it to be easily detected and imaged

• NB – If substrate is required, incubate the membrane again

after washing away the secondary antibody
1. Western blot using radioactive detection system
2. Protein ladder
3. Western blot using an antibody that recognizes
proteins modified with lipoic acid
 Clinical application

• HIV confirmatory test

• Testing for Lyme disease
• Confirmatory test for HBV
• Confirmatory test for HSV-2
• In anti-doping for then detection of rEPO
• Verification of protein production after cloning
 Advantages & Limitations

qAdvantages
• High sensitivity
• High specificity

qLimitations
• Costly
• Requires highly trained personnel
• Semi-quantitative
• Use of radioactive label is hazardous
• Time consuming