| Assay Method Information | |
| | BINDING INHIBITION ACTIVITY EVALUATION TEST |
| Description: | Coat the 96-well microplate (Costar) with 50 μL of recombinant human MAdCAM-1 (R&D Systems) solution per well, and incubate overnight (12-18 hours) at 4° C. Wash three times with 150 μL of TBS buffer per well. Block plate with 150 μL per well with blocking buffer for 1 h at 37° C. Wash three times with 150 μL of TBS buffer per well. Dilute the recombinant human integrin α4β7 (R&D Systems) with TBS buffer containing 0.1% bovine serum albumin (BSA), then add to the 96 well plate with 50 μL per well. Add 1 μL of test compound or DMSO, cover, incubate at room temperature for 2 h, wash three times with 150 μL of TBS buffer per well. Dilute the anti-β7 antibody (R&D Systems) with 0.1% BSA TBS buffer, then add to the 96 well plate with 50 μL per well, cover, incubate at room temperature for 1 h, wash three times with 150 μL of TBS buffer per well. Add 50 μL of streptavidin-HRP (R&D Systems) per well, incubate at room temperature for 20 min, wash three times with 150 μL of TBS buffer per well. Add 50 μL of TMB substrate solution (Sigma) per well, incubate at room temperature for 5-30 min, and add 25 μL of stop solution per well to stop the reaction. Finally, measure absorbance at 450 nm with microplate reader (SpectraMax 340PC. Molecular Devices). Repeat the test to find the binding rate of cells at each concentration when the absorbance of the well without the test substance is used as 100%, and calculate the concentration IC50 that causes 50% binding inhibition. |
| Affinity data for this assay | |
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