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

作者：Amini Nazila; Bayat Ali Ahmad; Zarei Omid; Hadavi Reza; Mahmoudian Jafar; Mahmoudi Ahmad R; Darzi Maryam; Rabbani Hodjattallah; Jeddi Tehrani Mahmood^*

来源：Protein and Peptide Letters, 2015, 22(5): 419-424.

DOI：10.2174/0929866522666141231111618

• Antibody
• beta-actin
• housekeeping protein
• peptide
• Western blot

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.

• 出版日期2015