Production of Polyclonal Antibodies for Detecting Human Type 2 Transglutaminase Variants in Colon Cancer

Abstract

Type 2 transglutaminase (TGM2) is a multifunctional ubiquitous protein, involving in protein cross-linking, apoptosis, and cell differentiation. Recently, some reports have suggested that TGM2 expression is a potential prognostic marker, and often associates with advanced stages of disease, metastatic spread, as well as drug resistance in many cancer cell lines although its primary function is unknown. To elucidate the role of TGM2 in cancer, the expression profile of the TGM2 need to be examined. In this study, new polyclonal antibodies detecting four TGM2 variants encoding protein from ENSEMBL database are produced and their specificities are confirmed by western blot analysis with E.coli overexpressing TGM2-002 protein, HEK293T cells overexpressing TGM2-S protein and human colon cancer samples. Western blot data showed that these antibodies could detect not only TGM2-002 in E. coli overexpressing TGM2-002 but also smaller molecular weight protein (about 62 kDa) in HEK293T overexpressing TGM2-S cells which was further confirmed by MALDI-TOF analysis. We found that both TGM2-1 and TGM2-4 antibodies could detect the full length TGM2-002 protein in colon cancer samples. Furthermore, in normal sample, we found that majority of TGM2-002 protein existed in membrane fraction but not in total lysate whereas TGM2-002 protein was found in both total lysate and membrane fraction in colon cancer samples.