Association of nonreceptor tyrosine kinase c-yes with tight junction protein occludin by coimmunoprecipitation assay.
Immunoprecipitation is one of the most commonly used techniques to study protein-protein interaction in vivo. There are three major steps involved in an immunoprecipitation procedure: 1) lysis of the cells to make the antigen of interest available; 2) formation of the antibody-antigen complex by adding specific antibody; 3) separation and detection of the immune complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Both polyclonal and monoclonal antibodies can be used to immunoprecipitate the antigen. Polyclonal antibodies usually bind to multiple sites on the antigen and, therefore, are more efficient than monoclonal antibodies. The advantage of using monoclonal antibodies for immunoprecipitations is the specificity of the antibody-antigen interaction because monoclonal antibodies bind to only one epitope of the antigen. If the antigen of interest is tyrosine phosphorylated, and this tyrosine phosphorylation is under investigation, each step has to be handled with extra care to ensure success of the procedure. The buffer used to lyse the cells depends on the nature of the antigen, with more stringent conditions required for integral membrane proteins than for soluble cytosolic proteins. In either case, it is very important to add the protease inhibitors to the lysis buffer in order to prevent degradation of the antigen.
Chen
Yan-Hua
YH
Department of Anatomy and Cell Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA.
Lu
Qun
Q
D004847:Epithelial Cells / Q000201:enzymology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin; D011233:Precipitin Tests / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051575:Proto-Oncogene Proteins c-yes; D029589:Seminal Plasma Proteins / Q000378:metabolism*; D019108:Tight Junctions / Q000201:enzymology*
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D029589:Seminal Plasma Proteins; D011505:Protein-Tyrosine Kinases; D051575:Proto-Oncogene Proteins c-yes; C489834:YES1 protein, human