Resolution of triplet components by electrophoresis
Following electrophoresis of platelet-extracted triplets obtained as
described above in 6% polyacrylamide gel tubes in Tris-glycine pH 8.3,
one gel was stained with Coomassie Brilliant blue G-250 and the bands of
O-glycoproteins, albumin and antibody, identified by electrophoretic
mobility of corresponding samples isolated from plasma [1], were cut
and electroeluted separately. The two glycoprotein bands were also
eluted together where required.
ELISA for ligand binding and inhibition assays Protein or
lectin was coated on Nunc MAXISORB microplate wells (maximum 1 µg in 200
µl PBS) by incubation at 37 °C for 3 h or overnight at 4 °C. Wells were
washed with PBS containing 0.05 % Tween-20 (PBST) and blocked by
incubation for 30 min at 37 °C with PBS containing 0.5 % Tween-20.
After another wash with PBST wells were incubated for 2 h at 4 °C with
200 µl PBST containing HRP-labeled or free primary ligands pre-incubated
with or without specific sugars. In cases where wells were incubated
with primary ligands, wells were washed and treated with HRP-labeled
secondary ligands. After washing again thrice with PBST plate-bound HRP
was measured by adding 200 µl OPD (0.5 mg/ml) in 0.1 M
citrate-phosphate buffer, pH 5.0 containing 0.03%
H2O2 for 15 min, followed by addition of
50 µl 12.5 % H2SO4 to stop the reaction
and absorbance measured at 490 nm in BIOTEK ELx800 ELISA reader.