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Production and Characterization of Polyclonal Antibody Against a Synthetic Peptide From β-actin Protein

Production and Characterization of Polyclonal Antibody Against a Synthetic Peptide From β-actin Protein

Nazila Amini, Mohadeseh Naghi Vishteh, Omid Zarei, Reza Hadavi, Negah Ahmadvand, Hodjattallah Rabbani, Mahmood Jeddi-Tehrani

Iran J Basic Med Sci. 2014 Jun;17(6):396-400.

PMID: 25140199

Abstract:

Objectives:
Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems.
Materials and methods:
A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH) and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples.
Results:
The antibody could recognize the immunizing peptide in ELISA. It could also recognize β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry.
Conclusion:
Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA, immunocytochemistry and immunohistochemistry.

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