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AAT Bioquest

What are the steps involved in western blot?

Posted October 25, 2022


Answer

There are a total of six steps involved in western blot.

  • In the first step, proteins are extracted from different samples such as cells or tissues, and broken down through sonication. The concentration of the extracted proteins is then taken to determine the mass of proteins loaded into each well.
  • In the second step, the proper SDS-PAGE gel must be chosen, as separating gels are made in 5%, 8%, 10%, 12%, or 15%. For proteins that have a lower mass, a higher percentage gel should be used.
  • In the third step, proteins are transferred electrophoretically to create an identical SDS-PAGE gel.  The proteins are moved from the gel onto a membrane (PVDF) to make proteins available for antibody detection. This process is known as electroblotting, and can occur in wet or semi-wet conditions. The membrane becomes sandwiched between the gel surface and filter.
  • The fourth step is the blocking of nonspecific protein binding sites on transfer membranes. Typically, BSA or nonfat dry milk are used as blocking agents. The proteins attach to all the places in the membrane where the target proteins have not attached. This is so the noise in the final product can be reduced and produce clear results.
  • The fifth step is the incubation of the membrane with a primary antibody specific to the epitope of interest. The primary antibody binds to the target protein when the primary antibody is incubated with the membrane. The membrane then becomes washed and becomes exposed to the enzyme conjugated secondary antibody. The labeled secondary antibody binds to the primary antibody which has already reacted with the target proteins.
  • The last step is the visualization of bound antibodies. A substrate reacts with the enzyme that is bound to the secondary antibody to create a colored substance. It can then be analyzed through different detection methods (colorimetric, chemiluminescent and fluorescent).
Additional resources

Western Blotting Assays

MegaWox™ polyHRP-Goat Anti-Mouse IgG Conjugate

Steps in Western Blotting