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Fig. 8. Identification of sperm surface WGA binding proteins. Sperm surface proteins were biotinylated, purified using streptavidin-conjugated magnetic beads and resolved using duplicate 2D SDS-PAGE. (A) The preparative gel was stained with Coomassie Brilliant Blue (G-250) and (B) the analytical gel was transferred to a nitrocellulose membrane and blotted with streptavidin HRP to assess the efficacy of removal of the biotin tag, before (C) being stripped and reprobed with biotinylated WGA. Proteins that cross-reacted with WGA were excised from the preparative gel and subjected to MALDI-ToF mass spectrometry. Three of the four predominant WGA-binding proteins were identified as platelet endothelial cell adhesion molecule-1 (125 kDa, pI 6.5; acc. no. P16284), Zinc-
2-glycoprotein (32 kDa, pI 5; acc. no. P25311) and prostate-specific antigen (28 kDa, pI 6; acc. no. AAA60193). A minimum of three matching peptides (bold and underlined) were used to assign an identity to each protein.
