• 16
    Nov
  • Acrosome Biogenesis in the Hamster: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3)

Antibody Preparation

To prepare monoclonal antibodies, BALB/c mice were immunized with 25-50 p.g of the purified ALM fraction emulsified in Freund’s adjuvant. Animals received two booster injections at 2-wk intervals followed by a final tail-vein injection of 5 |xg ALM suspended in PBS. After four days, animals were killed, and their splenocytes were fused with X63-Ag 8.653 myeloma cells (ATCC, Rockville, MD) using polyethylene glycol . Hybrid cells were selected by culture in RPMI 1640 HAT (hypoxanthine, aminopterin, thymidine) medium (Sigma Chemical Co.), and positive wells were identified by ELISA. Positive cells were cloned twice by limiting dilution and used to prepare ascites fluid .

Polyclonal antiserum against the 22-kDa ALM polypeptide (AM22) was prepared in guinea pigs. The ALM fraction was subjected to preparative SDS-PAGE , polypeptides were visualized by staining with CuCl2 , and the AM22 band was excised. Guinea pigs received a primary injection and two booster injections, at 2-wk intervals, of AM22 emulsified in Freund’s adjuvant. Either whole serum or an IgG fraction, purified by protein A Se-pharose affinity chromatography , was used for im-munocytochemistry or immunoblotting. flovent inhaler

SDS-PAGE, Western blotting, Peptide Mapping, and N-Terminus Sequencing

SDS-PAGE was performed on gradient or continuous gels as specified in the text. For peptide mapping, polypeptide bands were visualized by CuCl2 staining , excised, and equilibrated with 0.1% SDS, 1 mM EDTA, 2 mg/ml dithiothreitol (DTT), 20% glycerol, and 25 mM Tris-HCl, pH 6.8. The polypeptides were then fractionated on 15% SDS-polyacrylamide gels in the presence of V8 protease . Peptide fragments were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes and stained with Coomassie blue dye for N-terminus sequencing with an Applied Biosystems (Foster City, PA) 475S sequencer . Peptide sequences were analyzed with the National Center for Biotechnology Information (NCBI) BLAST program using the blastp database .

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