Western Blotting: Principle, Procedure and Applications

Definition: Technique for detecting specific proteins separated by electrophoresis by use of labelled antibodies.

• Devised by Towbin in 1979
• The term “Western” has no scientific significance just a misnomer.
• Principle: antigen antibody interaction, an immunodetection method

Summary of Procedure:
1. Extract and purify protein from cells
2. Separated by SDS-PAGE (Sodium dodecyl sulphate-PolyAcrylamide Gel Electrophoresis)
Function of SDS: SDS is an anionic detergent. Here it denatures protein and impart an overall negative charge. Therefore separation is based on size


3. Transfer proteins from gel to nitrocellulose paper
4. Blocking nonspecific antibody sites on the nitrocellulose paper with bovine serum albumin (BSA) or milk powder
5. Probing electroblotted proteins with primary antibody
6. Washing away nonspecifically bound primary antibody
7. Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate or formation of a diaminobenzidine (DAB) precipitate, radiolabelling or use of fluorescently labelled secondary antibody.
8. Autoradiography or photographing or fluorescence detection

Different samples of equal quantity are loaded in each lane. From the blot it is clear that protein expression (40,000 MW) is more in lane 4 and 5.
Applications:

• Highly sensitive method to detect a specific protein even in very low quantity.
• Used in clinical diagnosis
• Quantifying a gene product (gene expression studies)