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Assay Method Information

Assay Name:  Src IC50 Determination
Description:  IC50 values were determined with the Z lyte assay system (Invitrogen). The reactions were performed in 384-well small volume plates from Greiner (#784076). The kinase reaction for cSrc consisted of kinase buffer, kinase, peptide and ATP. For each IC50 determination, 16 different concentrations of inhibitor (range from 250,000 to 10 nM) were used in triplicate and at least three independent experiments were performed. Before starting the kinase reaction, enzyme and inhibitor were incubated for 40 min. Following the kinase reaction, Development Solution (included with the kit) was added to cleave the remaining unphosphorylated peptide. Following a 1 h incubation time with Development Solution, Stop Solution (included with the kit) was added to the reaction mixture. Fluorescence was then measured with a Spectramax M5 plate reader from Molecular Devices or a Safire2 from Tecan. Upon excitation of coumarin at 400 nm, fluorescence emission was measured at 445 nm (coumarin) and 520 nm (fluorescein). The ratio of coumarin to fluorescein emission was used to calculate the percentage of phosphorylation of the peptide by the kinase.
Affinity data for this assay
 

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Last update November 1, 2007
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