assay protocol

Protein identification:

Here we describe the tips for western blot after IP. In the last step of IP, after centrifuge you get antibody-agarose beads complex, thus in an eppendorf tube you have antibodies, target protein, protein A/G-beads and some non-specific binding proteins. Among antibodies, target protein and protein A/G-beads are non-covalent bonds. Only protein A/G covalently binds agarose beads.

When loading samples in SDS-PAGE, loading buffer (sample buffer) contains mercaptoethanol. After being heated all proteins are denatured. Protein A/G-beads are discarded by low-speed centrifuge. Then in your sample only target protein and its antibody are left. Because of the mercaptoethanol in loading buffer the disulfide bond between heavy chain and light chain is destructed and antibody molecule breaks into heavy chain molecule (55KD) and light chain molecule (25KD). Therefore, besides in final development target protein can be detected, if the secondary antibody in WB is from the same species as the primary antibody in IP, the heavy chain and the light chain can also be detected. Since the amount of antibodies applied is large (1μg), if molecular weight of target protein is close to that of heavy chain or light chain, the WB result will be fatally affected by signals of heavy chain and light chain.

To solve this problem, there are two methods:

  • 1. Choose antibodies from different species to perform western blot and immunoprecipitation respectively. Then use a secondary antibody that has weak species cross-reactivity or no species cross-reactivity in western blot. In this way signals of heavy chain and light chain are largely weakened.
  • 2. Use crosslinking agent to crosslink antibody and protein A/G-beads. Then by adding in loading buffer without mercaptoethanol and centrifuge, antibody and protein A/G-beads can be removed with only target protein left in the supernatant.