SDS 6-16% gradient polyacrylamide gels were used for protein separation, and iso-electrofocalization was performed as previously described. The gels were Coomassie blue-stained before spot cutting and mass-spectrometry analysis or silver stained when better visualization of the protein spots was needed. For protein quantification, the Coomassie blue-stained gels were scanned and the total protein content of each lane analyzed using the 1D-Elite software package (Amersham-Pharmacia Biotech AB, Uppsala, Sweden).
Semidry transfer of proteins was performed over 2 h at 0.8 mA/cm2. The western blots were blocked with TBS-Tween 20 (0.5%, w/v) and, depending on the antibodies used, supplemented with lyophilized low-fat milk (5% [w/v]), gelatin (0.5% [w/v]), or BSA (1% [w/v]).
The first antibodies were either rabbit polyclonals or mouse monoclo-nals. The anti-HE1, anti-RABP, anti-PGDS, and anti-acrosin are rabbit polyclonal antibodies obtained in our laboratory against the ram epididy-mal cholesterol transfer protein (CTP/HE1/NP-C2), retinoic acid binding protein (E-RABP), prostaglandin D2 synthase (PGDS), and sperm acrosin, respectively.