Characterization of the SPM Binding to the Carbohydrate Probes by Saccharide Competition Assay
Competition of SPM binding to the various prepared biotinylated carbohydrate conjugates was tested in polystyrene 96-well microtiter plates. The plates were coated with 0.5 |xg SPM protein/well diluted in coating buffer and plates were blocked as described for ELCBA. Various concentrations from 0 to 100 mM of inhibitory carbohydrate solutions in 50 |xl of incubation buffer were added per well for 1 h. Subsequently, the inhibition on immobilization of biotinylated carbohydrates was quantified as described above.
Staining of the Uncapacitated and Capacitated Sperm with Biotinylated Carbohydrate Probes
Sperm samples were washed twice in HBS containing BSA (HBS+B; 1% [w/v] BSA in HBS; centrifugation for 5 min at 300 X g) and 90-|xl aliquots (containing 20 X 106 sperm cells/ml) were incubated for 15 min at room temperature with the biotinylated carbohydrate-conjugate of choice by adding 10 |xl containing 1 mg/ml conjugate. Subsequently, 100 l^l of 5 ng/ml of streptavidin-fluorescein isothiocyanate (FITC) was added for 15 min.