All the cloned antiplatelet IgGs bind specifically to purified human talin and immunoprecipitate talin from platelet lysates. (A) Fab's were tested by ELISA for binding to talin purified from human platelets, the irrelevant antigen ovalbumin (OVA, control), and the flow-through preparation from the talin purification containing the talin-depleted platelet lysate. The anti-gp120 Fab b12 and secondary Ab alone were included as negative controls. (B) Fab's (5 μg) were incubated with platelet protein fractions enriched for talin, followed by capture of the ICs with goat anti-human IgG F(ab′)2 protein G-Sepharose. The immunoprecipitated proteins were separated by SDS-PAGE, blotted, and stained with a murine anti-talin-H mAb, TA205. Bands corresponding to intact talin (235 kDa) and talin-H domain (47 kDa) are identified. No talin staining was observed following immunoprecipitation using the anti-HIV-1 gp120 Fab b12 (negative control).
