American Society of Hematology

Fig. 5.

$Fig. 5. Affinity chromatography of whole-platelet lysates. / (A) SDS–5%-to-20% polyacrylamide gel of platelet lysate electrophoresed under reducing conditions and stained with Coomassie blue. (B-C) Protein from platelet lysate (A) that had bound to a control peptide column (B) or a GPIbβ (R149–L167) peptide-agarose column (B1 peptide) (C) was eluted with 10 mM EGTA in the washing buffer. Eluted 2-mL fractions1-4 were analyzed on SDS–5%-to-20% polyacrylamide gels under reducing conditions and stained with Coomassie blue. (D) Immunoblot with anticalmodulin monoclonal antibody of purified bovine calmodulin (CaM), the 22-kd protein isolated from platelet cytosol by EGTA elution from the GPIbβ peptide column (B1) or a GPV (K529–G544) peptide-agarose column (V). The proteins were electrophoresed on SDS–5%-to-20% polyacrylamide gels, electrotransferred to nitrocellulose, and visualized by means of a peroxidase-conjugated anti–mouse IgG and the ECL reagent.$

Affinity chromatography of whole-platelet lysates.

(A) SDS–5%-to-20% polyacrylamide gel of platelet lysate electrophoresed under reducing conditions and stained with Coomassie blue. (B-C) Protein from platelet lysate (A) that had bound to a control peptide column (B) or a GPIbβ (R149–L167) peptide-agarose column (B1 peptide) (C) was eluted with 10 mM EGTA in the washing buffer. Eluted 2-mL fractions1-4 were analyzed on SDS–5%-to-20% polyacrylamide gels under reducing conditions and stained with Coomassie blue. (D) Immunoblot with anticalmodulin monoclonal antibody of purified bovine calmodulin (CaM), the 22-kd protein isolated from platelet cytosol by EGTA elution from the GPIbβ peptide column (B1) or a GPV (K529–G544) peptide-agarose column (V). The proteins were electrophoresed on SDS–5%-to-20% polyacrylamide gels, electrotransferred to nitrocellulose, and visualized by means of a peroxidase-conjugated anti–mouse IgG and the ECL reagent.

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