VPB 321: Animal Biotechnology (2+1)

STEPS IN WESTERN BLOTTING

• A protein sample is subjected to poly acrylamide gel electrophoresis (PAGE).
• After this the gel is placed over a sheet of nitrocellulose and the protein in the gel is electrophoretically transferred to the nitrocellulose.
• The nitrocellulose is then soaked in blocking buffer (3% skimmed milk solution) to "block" the nonspecific binding of proteins.
• The nitrocellulose is then incubated with the specific antibody for the protein of interest.
• The nitrocellulose is then incubated with a second antibody, which is specific for the first antibody. For example, if the first antibody was raised in mouse, the second antibody might be termed "goat anti-mouse immunoglobulin”.
• The second antibody will typically have a covalently attached enzyme which, when provided with a chromogenic substrate, will cause a color reaction. Thus the molecular weight and amount of the desired protein can be characterized from a complex mixture (e.g. crude cell extract) of other proteins by Western blotting.