PKCalpha Human PKC Kinase Enzymatic Radiometric LeadHunter Assay [10 μM ATP] - TW

Procedure Summary:

Human recombinant protein kinase Ca expressed in Sf9 insect cells is used. Test compound and/or vehicle is preincubated with 100 ng/ml enzyme‡, 1.5 mM Ca2+, 100 μg/ml phosphatidylserine and 20 μg/ml diacylglycerol in modified MOPS buffer pH7.2 for 15 minutes at 25⁰C. The reaction is initiated by addition of 250 μg/ml histone H1, 10 μM ATP and 0.25 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]Histone H1 formed. Compounds are screened at 10 μM.

Substrate:

250 µg/ml Histone

ATP Concentration:

10 uM

Incubation:

10 min at 25°C

Control Inhibitor:

Staurosporine

Criteria For Significance:

≥ 50% of max stimulation or inhiibition

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:

2