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Publisher Summary This chapter discusses the purification of platelet activation-dependent granule to external membrane (PADGEM) glycoprotein. The enzyme-linked immunosorbent assay (ELISA) is primarily used for identifying murine antiplatelet antibodies with the desired properties. Parallel ELISAs are performed using either fixed gel-filtered thrombin-activated platelets or fixed resting platelets bound to microtiter wells to select antibodies specific only for the surface of activated platelets. Double-antibody suspension or fluid-phase radioimmunoassay is also used for the measurement of antiplatelet activation. The PADGEM protein is purified from crude platelet extracts by affinity chromatography using monoclonal antibody bound to a solid support. The KC4 antibody is purified from ascites fluid and then covalently linked to sepharose 4B. Sepharose is washed with cold phosphate-buffered saline, pH 7.4, and activated with 200 mg cyanogen bromide per mL Sepharose. The activated Sepharose is mixed with 25 mg of purified KC4 antibody in phosphate-buffered saline (PBS) and stirred overnight at 4°C. After washing, the KC4 antibody sepharose is packed into a column, equilibrated in PBS, and stored at 4°C for use in the affinity purification of the PADGEM protein. |
