A Novel Monoclonal Antibody Against a Synthetic Peptide from beta-Actin can React with its Corresponding Protein

Abstract

Actin is one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells with important roles in many cell functions. Antibodies against beta-actin and other housekeeping gene-encoded proteins are used as internal loading controls in Western blot analyses. The aim of this study was to produce a monoclonal antibody (mAb) against a synthetic peptide derived from N-terminal region of beta-actin and to study its reactivity with different organisms. A synthetic peptide, derived from beta-actin, was designed and used to produce a mAb by hybridoma technology. The produced antibody (clone 4E5-A10) was purified by an affinity chromatography column followed by characterization of purified mAb using SDS-PAGE, ELISA and Western blot. Our results showed that 4E5-A10 was an IgM and had desired purity and excellent reactivity with the immunizing peptide with an affinity constant of 2.7x10 8 M-1. It could detect a band of about 45 kDa, corresponding to beta-actin, in Western blot. Furthermore, it could react in a more sensitive manner and with a wider range of organisms than a known commercial anti beta-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of beta-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.