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Assay Method Information

Assay Name:  IGF-1R Kinase Biochemical Assay
Description:  Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 3 mM MnCl2, 1 mM DTT, 3 microM Na3VO4, and 0.2 mg/mL BSA. 3x KB is buffer of the same composition and pH as KB, but with three times the concentration of each component. Prior to starting the kinase inhibition assay, IGF-1R was pre-phosphorylated in order to linearize reaction kinetics. To achieve this, the desired total quantity of enzyme was prepared at an enzyme concentration of 360 nM in KB containing 100 microM ATP, and this preparation was incubated for 30 min at 28° C. 3x Enzyme Mix was obtained by diluting this preactivated enzyme 20-fold in 3x KB. The kinase assay was run with a final enzyme concentration of 6 nM, in the presence of 6 microM ATP, 1 nM 33P-γ-ATP and 10 microM substrate, a carboxy-terminally biotinylated peptide of the following sequence: KKKSPGEYVNIEFGGGGGK-biotin (SEQ ID NO:5). The peptide was obtained in batches of >95% peptide purity from American Peptide Company, Inc. (Sunnyvale, Calif., USA).
Affinity data for this assay
 

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Last update November 1, 2007
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