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Assay Method Information |
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| | Binding Assay |
| Description: | Fibrinogen was diluted with coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and coated with 100 uL/well to Nunc-Immuno maxisorp plates over night at 4 C. After discarding the coating solution plates were washed 3 times with buffer 1 (25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM MnCl2, 1 mg/ml BSA) and blocked with 100 uL blocking buffer (3% BSA in PBS 0.1% Tween20) for 1 hour at room temperature. After washing the blocked plates (3 times) with buffer 1, integrin alphaIIbbeta3 (50 uL) and either inhibitor (serial dilution in buffer 3, 25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM MnCl2, 1 mg/ml BSA 1 mM MgCl2, 1 mM CaCl2,) or buffer 3 (50 uL) were added to the wells and incubated for one hour at RT. Plates were then washed (3 times) with buffer 3 and incubated with 100 uL of anti-alphaIIbbeta3 antibody (anti CD41b, Pharmingen) in buffer 3 for 1 hour at RT. Plates were washed (3 times) with buffer 3 and incubated for 1 hour with 100 uL secondary antibody. |
| Affinity data for this assay | |
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