title: Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran ; Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Nazila
authors.initials: N
authors.lastname: Amini
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Mohadeseh Naghi
authors.initials: MN
authors.lastname: Vishteh
authors.affiliation: Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Omid
authors.initials: O
authors.lastname: Zarei
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Hadavi
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Negah
authors.initials: N
authors.lastname: Ahmadvand
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Hodjattallah
authors.initials: H
authors.lastname: Rabbani
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Mahmood
authors.initials: M
authors.lastname: Jeddi-Tehrani
keywords: Antibody; Immunocytochemistry; Immunohistochemistry; Peptide; Western blot; β-actin