A Time- and Cost-Saving Method of Producing Rat Polyclonal Antibodies

Abstract

Producing antibodies usually takes more than three months. In the present study, we introduce a faster way of producing polyclonal antibodies based on preparation of the recombinant oligopeptide as antigen followed by immunization of rats. Using this method, we produced antisera against two mouse proteins: ERGIC-53 and c-Kit. An expression vector ligated with a pair of complementary synthetic oligodeoxyribonucleotides encoding the protein was introduced into bacteria, and the recombinant oligopeptide fused with the carrier protein glutathione-S-transferase was purified. Wistar rats were immunized by injecting the emulsified antigen subcutaneously into the hind footpads, followed by a booster injection ­after 2 weeks. One week after the booster, the sera were collected and examined for the antibody titer by immunohistochemistry. Antisera with 1600-fold titer at the maximum were obtained for both antigens and confirmed for their specificity by Western blotting. Anti-­ERGIC-53 antisera recognized acinar cells in the sublingual gland, and anti-c-Kit antisera recognized spermatogenic and Leydig cells in the testis. These antisera were applicable to fluorescent double immunostaining with mouse monoclonal or rabbit polyclonal antibodies. Consequently, this method enabled us to produce specific rat polyclonal antisera available for immunohistochemistry in less than one month at a relatively low cost.

Expression and localization of receptor protein tyrosine phosphatase β and its ligand pleiotrophin in the submandibular gland of mice

“…Rat polyclonal antisera against the cytoplasmic domain of RPTPb-S (common with that of RPTPb-L) were produced in our laboratory according to the method described previously. 24,25 Briefly, a recombinant oligopeptide 41 amino acids in length corresponding to the Cterminus of RPTPb-S, that was fused with the carrier protein glutathione-s-transferase, was produced in bacteria BL21 (Novagen, Madison, WI) by introducing the expression vector pGEX-6p-1 (Amersham Pharmacia Biotech, Uppsala, Sweden). The oligopeptide was then emulsified with Freund's complete adjuvant and injected as antigen into footpads of adult female Wistar rats.…”

Expression, Localization, and Binding Activity of the Ezrin/Radixin/Moesin Proteins in the Mouse Testis

“…Rat monoclonal anti-radixin (clone R21) antibody was from Sanko Junyaku (Tokyo, Japan), and mouse monoclonal anti-a-tubulin (clone DM 1A) and anti-b-actin (clone AC-15) antibodies were from Sigma-Aldrich (St. Louis, MO). Rat polyclonal antisera against mouse radixin and moesin were produced in our laboratory according to the method de-scribed previously, with a modification (Wakayama et al 2006). Briefly, cDNA fragments 123 bp in length coding the carboxyl-termini of mouse radixin and moesin, respectively, were cloned into the bacterial expression vector pGEX-6p-1 (Amersham Pharmacia Biotech; Uppsala, Sweden).…”

Coexpression of Menin and JunD during the Duct Cell Differentiation in Mouse Submandibular Gland

“…Rat polyclonal anti-menin antibody was produced in our laboratory according to methods described previously (Wakayama et al 2006). Briefl y, a recombinant oligopeptide for menin, which was the C-terminal 39 amino acids of menin fused with the carrier protein glutathione-S-transferase, was produced in bacteria, emulsifi ed with adjuvant, and injected into the hind footpads of rats.…”