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Preparation of human GPIIb-IIIa.

By Dolores Lane,2014-06-22 13:39
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Preparation of human GPIIb-IIIa.

    1. GPIIb-IIIa was purified fromoutdated platelet concentrates using affinity

    chromatography.

    2. Briefly, human platelets suspended in Tris-buffered saline (TBS; 150

    mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 1 mM benzamidine (GP

    buffer) were sonicated and ultracentrifuged at 100,000g for 60 min at

    4?.

    3. The pellet was then resuspended in GP buffer containing 1% Triton

    X-100 (wt/vol) and allowed to stand on ice for 1 h.

    4. The solubilized platelet membrane preparation was ultracentrifuged

    again as indicated above.

    5. The platelet membrane lysate was applied to the affinity column, in

    which the IgG fraction of mAb to human GPIIb-IIIa was coupled to

    cyanogen bromide-activated SepharoseCL-4B (Amersham Pharmacia

    Biotech Inc., Piscataway, NJ).

    6. After extensive washing with 0.1% Triton X-100/GP buffer containing

    0.5 M LiCl, the bound GPIIb-IIIa was eluted with 0.05 M diethylamine in

    0.05% Triton X-100/GP buffer (pH 11.5).

    7. Fractions with a peak in the OD280 reading were collected as

    GPIIb-IIIa antigen. After GPIIb-IIIa was eluted, fractions in which the

    OD280 reading was below 0.1 were collected as a control preparation. 8. GPIIb-IIIa and control preparations were immediately neutralized with

    solid glycine, dialyzed against TBS containing 0.05% Triton X-100,

    sterilized by passage through 0.45μm pore-size syringe filters, and

    stored at-80?.

    9. The relative amounts of purified GPIIb-IIIa determined by Coomassie

    blue staining revealed that GPIIb and IIIa represented.

10. 95% of the total stained protein. GPIIb-IIIa was also purified from

platelets of two healthy donors on a small scale.

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