1. A Novel Monoclonal Antibody Against a Synthetic Peptide from β-Actin can React with its Corresponding Protein
- Author
-
Nazila, Amini, Ali-Ahmad, Bayat, Omid, Zarei, Reza, Hadavi, Jafar, Mahmoudian, Ahmad R, Mahmoudi, Maryam, Darzi, Hodjattallah, Rabbani, and Mahmood, Jeddi-Tehrani
- Subjects
Mice, Inbred BALB C ,Immunoglobulin M ,Blotting, Western ,Antibody Affinity ,Animals ,Antibodies, Monoclonal ,Electrophoresis, Polyacrylamide Gel ,Enzyme-Linked Immunosorbent Assay ,Female ,Amino Acid Sequence ,Peptides ,Actins ,Chromatography, Affinity - 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 2.7x10(8) 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.
- Published
- 2014