Research Article

Fast preparation of a polyclonal antibody against chicken protocadherin 1

Published: June 28, 2013
Genet. Mol. Res. 12 (2) : 2156-2166 DOI: 10.4238/2013.June.28.3

Abstract

Protocadherins constitute a large family belonging to the cadherin superfamily; they function in various tissues of a wide variety of multicellular organisms. However, their functions and expression modes are still unknown in many of these species and tissues. We developed a fast and low-cost method to produce polyclonal antibody against chicken protocadherin 1 (Pcdh1) that could be used in assays for immunological assessment of protein expression levels of chicken Pcdh1. Primers were designed with DNAStar, using the nuclear sequence of pcdh1 as a template; the pcdh101 fragment was amplified, identified by sequencing and cloned into expression vectors pGEX-2TK and pET-32a, separately, resulting in 2 recombinant plasmids, pGEX-2TK-pcdh101 and pET-32a-pcdh101. These were confirmed by double-enzyme digestion and sequencing. The recombinant expression vectors were transformed and expressed in Escherichia coli BL21. The recombinant oligopeptides glutathione-S-transferase (GST)-Pcdh101 and (His)6-Pcdh101 fused with the carrier protein GST and (His)6 separately, and were purified. Rats were immunized by injecting the emulsified GST-Pcdh101 antigen subcutaneously into their hind footpads, followed by a booster injection after 2 weeks. One week after the booster, the sera were collected and examined for antibody titer by indirect ELISA. The optimal dilution of this antiserum was 1:300. The specificity of the antiserum was confirmed by Western blotting. This antiserum had good specificity and could be used to detect chicken Pcdh1 in Western blot analysis. This method allows production of specific rat polyclonal antisera for Western blots in less than 1 month at a relatively low cost.

Protocadherins constitute a large family belonging to the cadherin superfamily; they function in various tissues of a wide variety of multicellular organisms. However, their functions and expression modes are still unknown in many of these species and tissues. We developed a fast and low-cost method to produce polyclonal antibody against chicken protocadherin 1 (Pcdh1) that could be used in assays for immunological assessment of protein expression levels of chicken Pcdh1. Primers were designed with DNAStar, using the nuclear sequence of pcdh1 as a template; the pcdh101 fragment was amplified, identified by sequencing and cloned into expression vectors pGEX-2TK and pET-32a, separately, resulting in 2 recombinant plasmids, pGEX-2TK-pcdh101 and pET-32a-pcdh101. These were confirmed by double-enzyme digestion and sequencing. The recombinant expression vectors were transformed and expressed in Escherichia coli BL21. The recombinant oligopeptides glutathione-S-transferase (GST)-Pcdh101 and (His)6-Pcdh101 fused with the carrier protein GST and (His)6 separately, and were purified. Rats were immunized by injecting the emulsified GST-Pcdh101 antigen subcutaneously into their hind footpads, followed by a booster injection after 2 weeks. One week after the booster, the sera were collected and examined for antibody titer by indirect ELISA. The optimal dilution of this antiserum was 1:300. The specificity of the antiserum was confirmed by Western blotting. This antiserum had good specificity and could be used to detect chicken Pcdh1 in Western blot analysis. This method allows production of specific rat polyclonal antisera for Western blots in less than 1 month at a relatively low cost.