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| | PTP1B Inhibition Assay |
| Description: | Steady-state kinetic parameters were measured for PTP1B using p-nitrophenyl phosphate (pNPP, 0.5−40 mM) as the substrate in NMR buffer. The rate of reaction was calculated by monitoring the change in absorbance at 405 nm where the molar absorbance for pNPP under these conditions is 12600 M−1 cm−1. Kinetic parameters were determined by fitting the reaction rates to different inhibition models as a function of the inhibitor and pNPP concentrations using GraphPad Prism version 6.0d. The total reaction volume in all cases was 500 μL, and the temperature was maintained at 25 °C using a water-regulated cuvette holder. The reaction was initiated by adding 10 μL of ∼25 μM protein for a final concentration of ∼0.5 μM PTP1B. The concentration of CGA was varied from 0.0 to 6.6 mM by adding 100 μL of serially diluted CGA in NMR buffer. The concentration of CGA was confirmed using the molar absorbance at 332 nm in ethanol (20030 M−1 cm&# |
| Affinity data for this assay | |
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