Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(1)

METHODS(1)

Materials

Unless otherwise stated, all routine chemicals were supplied by Sigma-Aldrich (Poole, UK) or Merck (Poole, UK). ‘‘Immobilon-P’’ PvDf membrane was supplied by Millipore (Watford, UK) and synthetic oligonucleotides by Sigma Genosys (Pampisford, UK). Culture supernatants containing McAbs to rat sperm membrane antigens 2D6 and 2B1, together with rabbit polyclonal antibodies (PAbs) to PBP and 2D6 antigens, were prepared and used as described previously. The 2D6 PAb was raised against the 24-kDa form of the protein by electroeluting the antigen from SDS gels. A guinea pig PAb to rat CD52 glycoprotein was a generous gift of Dr. Christianne Kirchhoff (Institute for Hormone and Fertility Research, Hamburg, Germany).

FITC-conjugated rabbit anti-mouse IgG (FITC-RAM), peroxidase-conjugated rabbit anti-mouse IgG (Px-RAM), FITC-conjugated sheep anti-rabbit IgG (FITC-SAR), and peroxidase-conjugated sheep anti-rabbit IgG (Px-SAR) were purchased from DAKO Ltd (Ely, UK). Rats (Wistar strain) and mice (Balb/c strain) were supplied by the small-animal facility at the Babraham Institute and hamsters by Harlan U.K. Ltd. (East Sussex, UK). Ejaculated ram, bull, and boar semen were collected from animals maintained at the Institute using an artificial vagina. A cDNA library from whole rat epididymis was custom-made in pSPORT 1 vector by the Deutsches Ressourcenzentrum fur Genomforschung GmbH (RZPD; Berlin, Germany). 4,2-Aminoethylbenzenesulfonyl-fluoride (AEBSF or ‘‘Pefabloc’’; Melford Ltd, Ipswich, UK) was used as a protease inhibitor.

Category: Plasma

Tags: epididymis, gamete biology, male reproductive tract, sperm