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.