Anti cd42b

GST or GST-BR fusion proteins were diluted to 500 nM in PBS and 50 μL applied to microtiter wells. Plates were incubated 18 hr at 4°C. Unbound proteins were removed by aspiration, and wells were rinsed with 100 μL TEN buffer (10 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 8). An extract enriched for glycocalicin (the soluble extracellular domain of GPIbα) was prepared by sonication of washed human platelets, followed by incubation at 37°C for 1 h to allow the proteolytic cleavage of GPIbα and release of glycocalicin, as described.^{28 (link)} Insoluble debris was removed by centrifugation, followed by passage of the platelet extracts through a 0.45 μm filter. The platelet proteins were diluted to 60 μg/mL in TEN buffer containing Complete protease inhibitors (Roche) and 1X Blocking Reagent (Roche), with subsequent four-fold dilutions in the same buffer. Diluted platelet lysates (50 μL) were added to wells, and plates were incubated at 23 °C for 1 h with vigorous rocking. Wells were washed three times with PBS, and bound GPIbα was detected by ELISA, using a rabbit monoclonal anti-CD42b (abcam), followed by a peroxidase-conjugated goat anti-rabbit IgG (Sigma), and colorimetric detection with o-phenylenediamine (Sigma). The negative control was background subtracted from the measurements.