Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | Enzyme Inhibition Assay (IC50) and Fluorescence Quench Assay (KD) |
| Description: | Inhibitors were assayed in a 96-well plate format. Typically, protease was preincubated with concentrations of test compounds at 37 deg C for 30 min. The reaction was initiated by the addition of substrate Bz-nKRR-AMC. Reaction progress was monitored continuously by following the increase in fluorescence on a Tecan Safire2 plate reader. IC50 values of inhibitors were derived by fitting the calculated initial velocities to a nonlinear regression curve using GraphPad Prism software. Each point of the IC50 curve was measured in duplicate during a single experiment. Compounds were tested for binding (KD) using fluorescence quench assay. Two protein solutions were prepared (2-5 uM protein with and without 40 uM compound) and were mixed in a microplate to obtain 12 different compound concentrations ranging from 0 to 40 uM. Then each dilution comprising 90 ul was transferred to a UV-Star 96-well microplate. After 1 h incubation at room temperature, fluorescence was measured at 25 deg C on a Tecan Safire2 with ex = 280 nm and em = 340 nm. At the end of the measurements, 11 uM bovine pancreatic trypsin inhibitor (BPTI) was added to the wells containing 40 uM compound and the fluorescence was measured again. Binding curves were analyzed according to the two-state model describing the formation of a 1:1 complex. Kd values were obtained from curve-fit of binding isotherms. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |
|
Home |
| |
Search |
| |
Deposit |
| |
SiteMap |
| |
About us |
| |
Email us |
| |
Info |
|
©2000 BindingDB. All rights reserved. |
|||||||||||||