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

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

Volume: 22 Issue: 5

Author(s): Nazila Amini, Ali-Ahmad Bayat, Omid Zarei, Reza Hadavi, Jafar Mahmoudian, Ahmad R. Mahmoudi, Maryam Darzi, Hodjattallah Rabbani and Mahmood Jeddi-Tehrani

Affiliation:

Keywords: Antibody, β-actin, housekeeping protein, peptide, Western blot.

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 β-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 β-actin and to study its reactivity with different organisms. A synthetic peptide, derived from β-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 2.7x10⁸ M^-1>. It could detect a band of about 45 kDa, corresponding to β-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 β-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of β-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.

Cite this article as:

Amini Nazila, Bayat Ali-Ahmad, Zarei Omid, Hadavi Reza, Mahmoudian Jafar, Mahmoudi R. Ahmad, Darzi Maryam, Rabbani Hodjattallah and Jeddi-Tehrani Mahmood, A Novel Monoclonal Antibody Against a Synthetic Peptide from β-Actin can React with its Corresponding Protein, Protein & Peptide Letters 2015; 22 (5) . https://dx.doi.org/10.2174/0929866522666141231111618