Question

Investigators are studying a protein that must be phosphorylated to be activated. Which method could be used to separate the phosphorylated form of the protein from the form that lacks an activating phosphate group?

Answer 1

There is two common methods to separate a phosphorylated protein from the proteins who does not have the phosphate group.

1. Western Blot test

The Western blot test is a method where we separate of the biological sample with SDS- PAGE (electrophoresis method) and then we transfer to a nitrocellulose membrane. We use a phospho-specific antibody to identify the protein of interest.

2. The ELISA test

this test is also a powerful method for measuring protein phosphorylation. Elisa test is more are quantitative than Western blot test and also shows great advantage in studies related with kinase activity. In this test we use a capture antibody specific for the desired protein, independent of the phosphorylation state. The target protein is binded to the antibody-coated plate. and finally a detection antibody specific for the phosphorylation site analyzed is added. In these test it is possible to use colorimetric or fluorometric detection.