| Assay Method Information | |
| | Surface Plasmon Resonance (SPR) Assay |
| Description: | To establish binding constants for the compounds/drugs to PAI-1, an indirect approach using surface plasmon resonance (SPR) was employed. Varying concentrations of each compound were preincubated with PAI-1 in solution and then passed over immobilized anhydrotrypsin (Molecular Innovations) using a Biacore 2000 optical biosensor, and the loss of PAI-1 binding to anhydrotrypsin was quantified. Bovine anhydrotrypsin was immobilized to CM5 SPR chips at the levels of ~2000 response unit (RU) in 10 mM sodium acetate, pH 5.0. The reference flow cell surface was left blank to serve as a control. Remaining binding sites were blocked by 1 M ethanolamine, pH 8.5. All binding reactions were carried out in assay buffer. PAI-1 at 2 nM was first incubated with the indicated concentrations of inhibitor in running buffer for at least 15 min at 23 C.The slope of the association phase of PAI-1 binding to an immobilized ligand (e.g., vitronectin or anhydrotrypsin) has been shown to have a linear relationship. |
| Affinity data for this assay | |
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