VMC 221: Veterinary Immunology and Serology (1+1)

WESTERN BLOTTING (IMMUNOBLOTTING)

• Used to identify a specific protein in a complex mixture of proteins.
• It is named for its similarity to Southern blotting which detects DNA fragments and Northern blotting which detects mRNAs.
• In Western blotting, a protein mixture is electrophorectically separated on an SDS-polyacrylamide gel (SDS-PAGE).
• The protein bands are transferred to a nylon /nitrocellulose membrane passively or by electrophoresis and individual protein bands are identified by flooding the membrane with radiolabeled or enzyme linked antibody specific for the protein of interest.
• The antigen-antibody complexes that are formed (on the band containing the protein recognized by the antibody) can be visualized by a variety of methods (figure).
• If a radiolabeled antibody is used, the position of the protein band can be determined by exposing the membrane to an X ray film (this is called autoradiography).
• But usually enzyme labeled antibodies are used rather than radiolabeled ones.
• After binding of the enzyme tagged antibody to protein band of the membrane, addition of a chromogenic substrate that produces a colored and insoluble product causes the appearance of a colored band at the site of the target antigen/protein(Western blotting can also be used to identify a specific antibody in a mixture).