UIC-2011-128 – Human Carboxypeptidase D (CPD) Rabbit Polyclonal Antiserum

Carboxypeptidase D (aka: CPD, Metallocarboxypeptidase D, gp180), the human homolog of duck gp180, is a member of the regulatory B-type carboxypeptidase family. Carboxypeptidases perform a wide variety of roles, ranging from food digestion to the selective biosynthesis of hormones and neuropeptides. Unlike other metallocarboxypeptidases, CPD is broadly distributed among mammalian tissues where it is enriched in the trans Golgi network, and is also the only transmembrane metallocarboxypeptidase. As a result of its broad distribution, subcellular localization, and specificity for C-terminal Lys and Arg residues, CPD is thought to function following the action of endopeptidases in the processing of proteins that transit the secretory pathway, such as the precursors for neuroendocrine peptides, growth factors, and some growth factor receptors. A rabbit was immunized subcutaneously with a purified recombinant 20 kDa C-terminal fragment of human CPD emulsified in Freund’s complete adjuvant. After 2 weeks, the rabbit was boosted intramuscularly with the same amount of antigen in Freund’s incomplete adjuvant and subsequently every month, and was bled from a marginal ear vein 7-10 days after each boost. In order to affinity purify the antiserum, the recombinant protein was run in an SDS-polyacrylamide gel, blotted to a PVDF membrane, and the strip containing the 20 kDa band was excised. The membrane strip was incubated with antiserum to the 20 kDa fragment of human CPD. The bound antibody was eluted with 0.1 M glycine-HCl, pH 2.5, immediately neutralized with 1 M Tris-HCI, pH 8 and then diluted 1:100 for use in subsequent experiments. Veronica Haywood vhaywo2@uic.otm.edu 312-996-4865