The pronounced effects of disulfide bond cleavage on the size of the proteins recognized by 2D6 McAb suggests that they may be secreted as a multimeric complex and are progressively ‘‘processed’’ by reduction and endoproteo-lysis as they pass through the distal regions of the duct. Further evidence to support this interpretation is shown by Western blots taken from two-dimensional nonreducing/reducing SDS gels of cauda sperm proteins (Fig. 4, A and B). Before reduction the major protein component identified by 2D6 McAb had a molecular mass of 48 kDa, whereas after reduction it was displaced from the diagonal and migrated at 24 kDa. In addition, when the reduced form of 2D6 antigen (24 kDa) from cauda spermatozoa was electroeluted from SDS gels, incubated at 4°C for 2 days in 40 mM Tris-glycine buffer (pH 8.2), and reanalyzed ±2 mM DTT, it migrated at —250 kDa (Fig. 4C). Intermediate-size forms were not detected. This suggests that the 24-kDa antigen has strong tendencies to self-associate. yasmin side effects
In the course of this work it was found that the method of collection of CEP had a significant influence of its composition. When luminal fluid was collected from the cauda epididymidis by making three to four small incisions into the tissue with iridectomy scissors, nonreducing SDS-PAGE analysis of proteins in the CEP revealed two strongly staining proteins at 160 and 48 kDa that were only weakly present in CEP collected by micropuncture (results not shown). In addition, a diffusely staining protein at ~22 kDa was detected in CEP from minced tissue that was absent from micropuncture samples. This suggests that caution should be exercised in collecting CEP by uncontrolled mincing of the tissue.
FIG. 4. Nonreducing-reducing SDS-PAGE gels of detergent-solubilized proteins from cauda spermatozoa stained with (A) Coomassie blue or (B) Western blotted and probed with 2D6 McAb. Proteins were separated in the first dimension under nonreducing conditions, the gel strip was excised, and the contained proteins were reduced in situ with DTT followed by electrophoresis in the second dimension at 90° to the first direction. Arrow indicates position of the 24-kDa component. C) SDS-PAGE of the electroeluted 24-kDa component after incubation in buffer at pH 8.2 for 2 days under nonreducing (lane 1) and reducing (lane 2) conditions. The protein shows strong tendencies to self-associate.