| Assay Method Information | |
| | Binding Assay |
| Description: | Fibronectin 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 (Nalge Nunc Europe Ltd) 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 (50 uL) and either inhibitor (serial dilution in buffer 1) or buffer 1 (50 uL) were added to the wells and incubated for one hour at RT. Plates were then washed (3 times) with buffer 1 and incubated with 100 uL of anti-human-Fc-HRP antibody conjugate (Sigma-Aldrich, Taufkirchen, Germany) in buffer 1 for 1 hour at RT. After additional washing steps (3 times) with buffer 150 uL of HRP substrate solution TMB (Seramun, Germany) were added to the wells. |
| Affinity data for this assay | |
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