Aurora-B Human Other Protein Kinase Enzymatic Radiometric LeadHunter Assay [10 μM ATP] - TW

Procedure Summary:

Human recombinant protein kinase Aurora-B expressed in insect S21 cells is used. Test compound is preincubated with 4.2 μg/ml enzyme‡ in modified MOPS buffer pH 7.2 for 15 minutes at 37°C. The reaction is initiated by addition of 100 μM kemptide, 10 μM ATP containing 0.05 μCi[γ³²P]ATP for another 30 minute incubation period and terminated by further addition of 3% H3PO4. An aliquot is removed and counted to determine the amount of [32P]Kemptide. Compounds are screened at 10 μM.

Substrate:

100 µM Kemptide

ATP Concentration:

10 uM

Incubation:

30 min at 37°C

Control Inhibitor:

Staurosporine

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

10