2. • The process of transferring macromolecules from gel to a
membrane followed by their detection on the membrane is known
as Blotting
• DNA – southern blotting
RNA – northern blotting
Proteins – western blotting
• Western blotting allows investigators to determine molecular weight
of protein and to measure relative concentration of protein present
in different samples.
3. • Western blotting technique rely on the specificity of binding
between a molecule of interest and a probe to allow detection of
molecules of interest in a mixture of similar molecules .
• It is used for the identification of particular protein from the
mixture of protein .
• In this method labeled antibody against particular protein is used
to identify desired protein , so it is a specific test .
5. • Tissue preparation
• Gel electrophoresis
• Transfer
• Blocking
• Antibody probing
• Detection
• Analysis
6. TISSUE PREPARATION
• Protein is extracted from cell by mechanical or chemical lysis
• To prevent the digestion of the sample by its own enzyme –anti protease
and phosphatase are used
GEL ELECTROPHORESIS
• Separation of proteins
• SDS-PAGE allows separation of proteins according to molecular weight
• Sample to be analyzed is mixed with SDS.
• SDS is an anionic detergent , imparts negative charge to proteins.
• Polyacrylamide gel
• Buffer used -TRIS
7. TRANSFER
• Following gel electrophoresis, the separated proteins are transferred
from polyacrylamide gel to the membrane.
• Membrane : nitrocellulose or polyvinylidenedifluoride (PVDF)
• The membrane is placed on top of the gel , sandwiched in transfer stack
therefore when protein binds to these membranes , their epitopes are
easily accessible to antibodies.
• Methods : capillary
electrophoretic
• Proteins in the gel are negatively charged ,they move from the gel to the
membrane and becomes attached to it .
BLOCKING
• To prevent non-specific binding of antibodies .
• Antibodies are also proteins so they are likely to bind to nitrocellulose .
• It is essential to block spaces not occupied by proteins.
• Blocking agents used : bovine serum albumin (BSA) or casein
8. ANTIBODY PROBING
The specificity of antigen-antibody interaction permits the
identification of proteins in the sample
1. Primary
• Membrane is incubated with primary antibody .
• The antibody is specific to target protein .
• It binds to the target proteins on the membrane forming
antigen: antibody complex and the excess is washed off .
2. Secondary
• After washing ,the membrane is incubated with secondary
antibody to maximize sensitivity of detection .
• Binding of multiple secondary antibodies to the primary
antibody leads to amplification of detection signals.
• The excess secondary antibody is washed off.
9. DETECTION
• A substrate reacts with the enzyme that is bound to secondary
antibody to generate a coloured band .
• Enzyme – alkaline phosphatase or horseradish peroxidase(HRP)
it generates chemiluminescent signals upon addition of chromogenic
agent .
• The enzyme in the membrane catalysis oxidation of substrate into
an insoluble purple product.
10.
11. • SEPARATION
Proteins are separated by gel electrophoresis, usually sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE).
• TRANSFER
The proteins are transferred to a sheet of special blotting paper called
nitrocellulose or polyvinylidenedifluoride (PVDF).
• PROBING
The blot is incubated with antibody to bind to any remaining sticky places on
nitrocellulose .Secondary antibody is then added to the solution which is
able to bind to its specific protein .The antibody has an enzyme ( e.g. alkaline
phosphatase or horseradish peroxidase ) or dye attached to it .
• DETECTION
The location of antibody is revealed by incubating it with a colorless
substrate that the attached enzyme converts to colored product which can
be seen and photographed.
12. - To determine size and amount of protein in given sample.
- Disease diagnosis : detects antibody against virus or bacteria in
serum .
- Western blotting technique is the confirmatory test for HIV.
It detects anti HIV antibody in patient's serum.
- Useful to detect defective proteins . (prions disease)
- Definitive test for Creutzfeldt-Jakob disease , Lyme disease,
Hepatitis B.
13. Western blot for HIV:-
• Western blot is the highly specific confirmatory test for diagnosis
of HIV .
• The basic technique for WB involves sorting of proteins on gel
followed by its transfer and then probing with antibodies which
react with proteins that are being searched for.
For HIV western blot ,protein (antigen) is known ,they
look for antibodies in patients blood that stick to them.
• Antibodies against the following HIV proteins-
envelope: gp41 and gp 120
core : p17,p24, p55
• Proteins from known HIV –infected cells are separated and blotted
on membrane ,the serum to be tested is applied in the primary
antibody incubation step ,after washing the excess secondary
antibody conjugated with an enzyme is added. The stained band
will indicate whether the patient ‘s serum contains anti-HIV
antibody.
14. IMMUNOLOGY AND IMMUNOTECHNOLOGY
- ASHIM K. CHAKRAVARTY
IMMUNOLOGY - Dr. P MADHAVEE LATHA
https://www.onlinebiologynotes.com/western-blotting-
technique-principle-procedure-application/
https://novowb.wordpress.com/category/western-blot-
applications/
https://www.sigmaaldrich.com/technical-
documents/articles/biology/western-blotting-introduction.html
https://www.bosterbio.com/protocol-and-troubleshooting/western-blot-
principle
