Fig. 2. Comparison of the WGA-induced phosphotyrosine profile. Purified human spermatozoa were incubated in either BWW alone (BWW), BWW supplemented with 0.5 µg/ml WGA (WGA), or in positive (+ve) and negative (–ve) control media for 30 minutes. Following incubation, spermatozoa were solubilized in either CHAPS (1 hour on ice) or SDS extraction buffers (5 minutes, 100°C) and extracted proteins were prepared for immunodetection of phosphotyrosine residues (anti-py). The SDS-extract was characterized by the presence of two predominant proteins (83 and 105 kDa) however, overexposure of this blot (WGA*) demonstrated a pattern that approximated that seen in CHAPS extracts. This comparison was replicated three times with pooled semen samples obtained from at least three different donors, and representative blots are depicted.