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Epitope Analysis of PPARgamma Monoclonal Antibody Pgamma48.34A and Its Application for Screening PPARgamma Ligands

Epitope Analysis of PPARgamma Monoclonal Antibody Pgamma48.34A and Its Application for Screening PPARgamma Ligands

Hae-Sook Lee, Min-Chul Cho, Tae-Woong Baek, Yong-Kyung Choe, Jong-Wan Kim, Jin-Tae Hong, Pyung-Keun Myung, Sang-Gi Paik, Do-Young Yoon

J Immunol Methods. 2005 Jan;296(1-2):125-34.

PMID: 15680157

Abstract:

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that directly modulate gene expression by binding to specific ligands. It has been established that PPARgamma ligands play an essential role in obesity, diabetes, and inflammation. Recently, a great deal of research has focused on the screening of PPARgamma ligands. In this study, both a human peroxisome proliferator-activated receptors gamma2 (PPARgamma2) recombinant protein and a specific monoclonal antibody against PPARgamma2 were produced in order to screen PPARgamma ligands. Analysis of deletion mutants revealed that monoclonal anti-PPARgamma antibody Pgamma48.34A possesses an antigenic determinant in the N-terminal region (31-84 a.a) of human PPARgamma2. The results of Western blot testing revealed that Pgamma48.34A recognized both glutathione S-transferase (GST)- and his-tagged human and mouse PPARgamma recombinant proteins and also identified PPARgamma in adipocytes and mouse tissues. Compared to some commercially available antibodies, this antibody does not bind with skimmed milk or BSA and exhibits a higher degree of specificity. An in vitro binding assay revealed that PPARgamma2 was bound to steroid receptor coactivator-1 (SRC-1) in a dose-responsive manner in the presence of indomethacin, and Pr48.34A was able to detect PPARgamma in a complex consisting of PPARgamma and SRC-1. Using Pgamma48.34A antibody, an enzyme-linked immunosorbent assay (ELISA) system based on the binding between fPPARgamma2 and SRC-1 has been optimized to screen new PPARgamma ligands. This new antibody, Pgamma48.34A, exhibits higher degrees of both specificity and sensitivity against PPARgamma than do other commercial anti-PPARgamma antibody, and may constitute a profound contribution to the screening of PPARgamma ligands as well as the functional study of PPARgamma.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411932	SRC, active, GST tagged human			Price
IAR42414952	SRC1 (627-786),His tagged human			Price

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