SDS electrophoresis, Western blotting and lectin staining of platelet surface proteins
Platelets from normal blood (10 ml samples) separated from other cells by centrifugation at  4530 g as described earlier was lysed in cold hypotonic buffer (7.5 mM phosphate buffer pH 7.3 containing 10 mM EDTA; 600 µl). The lysate was centrifuged at 100,000 g for 1 h at 4°C to get a fraction enriched in platelet surface proteins as sediment. SDS polyacrylamide slab gel electrophoresis (7.5%) of this sample (1 mg/ml) was done as described by Laemmli [14]. Separated proteins were transferred to polyvinyl difluoride (PVDF) membrane by electroblotting as described by Towbin et al. [15].  The strips of 3-4 mm width were cut out from transfer membrane, blocked with PBS containing 0.2% Tween 20 and treated with HRP-conjugated lectin with or without specific sugar for 2 h at 4°C. The strips were then washed twice with 0.05 % PBST and once with PBS alone and stained using freshly prepared 4-chloronaphthol solution. As the protein bands appeared, strips were washed in PBS.