Preparation of P12 and Its Derivatives
The seminal vesicles of mature male mice (ICR, 8-12 wk; Charles River Laboratories, Wilmington, MA) killed by cervical dislocation were carefully dissected free of the adjacent coagulating glands. The secretions collected from 100 mice were expressed directly into 100 ml of ice-cold 5% acetic acid. P12 was purified from SVS according to our previous procedure. A fusion protein of GST-P12 was prepared from the expression of a recombinant DNA in E. coli. A recombinant P12 with the full activity of the naturally occurring P12, with regard to its inhibitory effect on trypsin activity, was recovered from the chimerical polypeptide. www.my canadian pharmacy.com
Detection of P12 and Its mRNA in Reproductive Tracts
The protein in normal saline (1.0 mg/ml) was mixed with an equal volume of complete adjuvant. Rabbits received intrasplenic injection of 0.25 ml of the mixture. After 12 wk, the animals were treated in the same way with 100 ^g of antigen in a mixture of normal saline and incomplete adjuvant (1:1 by volume). The antiserum was collected 2 wk later. The P12 antibody was partially purified by affinity chromatography on a protein A-Sepharose (Pharmacia, Uppsala, Sweden) column.