| Assay Method Information | |
| | CDK4/Cyclin D1 CHEF Assay |
| Description: | The purpose of CDK4/Cyclin D1 assay is to evaluate the inhibition (% inhibition and IC50 values) of small molecule inhibitors by using a Chelation-Enhance Fluorescence (CHEF) assay. In a CHEF assay, phosphorylation of a peptide substrate results in proportional increase in fluorescence. CHEF kinase assay use peptide substrates containing a synthetic alpha-amino acid with a side chain bearing an 8-hydroxyquinoline derivative (sulfonamido-oxide, Sox). Upon phosphorylation of a nearby serine, threonine or tyrosine and in the presence of Mg(II), the spectral properties of the Sox residue are altered, emitting 485 nm wavelength light when excited with a 360 nm wavelength light source. CDK4/Cyclin D1 catalyzes the phosphoryl transfer to the SOX-labeled substrate peptide AQT0258 from Assayquant Technologies. Typical reaction solutions contained 2% DMSO (+/− inhibitor), 10 mM MgCl2, 1 mM DTT, 200 uM ATP (ATP Km=195.2 uM), 0.012% Brig-35, 10 uM AQT0258 peptide, 0.02% BSA, 1% Glycerol, 0.55 mM EGTA, 2.5 nM CDK4/Cyclin D1 in 54 mM HEPES buffer at pH 7.5. The reaction was initiated with the addition of substrate solution, following a 30-minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. Reactions were allowed to proceed for 3 hrs at 22° C., followed by fluorescence read of the reaction. The IC50 determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the 4 parameters IC50 equation. |
| Affinity data for this assay | |
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