| Assay Method Information | |
| | Protein kinase assay (Condition B: Thiol-free Conditions) |
| Description: | The IC50 profile of compounds was determined using one protein kinase in a customized, thiol free assay. IC50 values were measured by testing 10 concentrations (1×10−05 M to 3×10−10 M) of each test compound in singlicate against each kinase of interest. Prior to testing, the 1×10−03 M stock solutions in column 2 of the master plates were subjected to a serial, semi-logarithmic dilution using 100% DMSO as a solvent. This resulted in 10 distinct concentrations, with a dilution endpoint of 3×10−08 M/100% DMSO in column 12. Column 1 and 7 were filled with 100% DMSO as controls. Subsequently, 2×10 microliter from each well of the serial diluted copy plates were aliquoted with a 96 channel pipettor into two identical sets of compound dilution plates . All plates were barcoded for automated identification and tracking purposes. IC50 values were measured by testing 10 concentrations (1×10−05 M to 3×10−10 M) of each compound in singlicate. All compounds were stored as powder until being solubilized in DMSO. Solubilized compounds were stored as 1×10−02 M/100% DMSO stock solutions. Prior to the assay process, 90 microliters of H2O were added to each well of a set of compound dilution plates. To minimize potential precipitation, the H2O was added to each plate only a few minutes before the transfer of the compound solutions into the assay plates. Each plate was shaken thoroughly, resulting in compound dilution plates with a final of 10% DMSO. For each assay, 5 microliters of solution from each well of the compound dilution plates/10% DMSO were transferred into the assay plate. The final volume of the assay was 50 μl. All compounds were tested at 10 final assay concentrations in the range from 1×10−05 M to 3×10−10 M, in singlicate. The final DMSO concentration in the reaction cocktails was 1% in all cases. A radiometric protein kinase assay (33PanQinase Activity Assay) was used for measuring the kinase activity of the protein kinase. All kinase assays were performed in 96-well FlashPlates from PerkinElmer (Boston, Mass., USA) in a 50 microliter reaction volume. The reaction cocktail was pipetted in four steps in the following order: 20 microliter of assay buffer (standard buffer) 5 microliter of ATP solution (in H2O) 5 microliter of test compound (in 10% DMSO) 20 microliter enzyme/substrate mix. Each assay for the protein kinase contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 microM Na-orthovanadate, 1 mM TCEP, 50 μg/ml PEG20000, ATP (corresponding to the apparent ATP-Km of the kinase, see Table A), [gamma-33P]-ATP (approx. 6×10×E5 cpm per well), with the protein kinase and relevant substrate being used in pre-determined amounts, depending on the kinase in question. For all experiments labeled as Thiol-free , all glutathione was exchanged from protein preparations so as to be removed from the assay and final buffer conditions contained no thiol-containing reagents. This was done so there would be no interference with the key cysteines in the proteins of interest. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |