title: A Novel Monoclonal Antibody Against a Synthetic Peptide from β-Actin can React with its Corresponding Protein.
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.
authors.forename: Nazila
authors.initials: N
authors.lastname: Amini
authors.forename: Ali-Ahmad
authors.initials: AA
authors.lastname: Bayat
authors.forename: Omid
authors.initials: O
authors.lastname: Zarei
authors.forename: Reza
authors.initials: R
authors.lastname: Hadavi
authors.forename: Jafar
authors.initials: J
authors.lastname: Mahmoudian
authors.forename: Ahmad R
authors.initials: AR
authors.lastname: Mahmoudi
authors.forename: Maryam
authors.initials: M
authors.lastname: Darzi
authors.forename: Hodjattallah
authors.initials: H
authors.lastname: Rabbani
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran. mahjed@avicenna.ac.ir.
authors.forename: Mahmood
authors.initials: M
authors.lastname: Jeddi-Tehrani
mesh_terms: D000199:Actins / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007075:Immunoglobulin M / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000737:chemistry / Q000276:immunology*
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D010455:Peptides