PKCbeta (2) Human PKC Kinase Enzymatic Radiometric LeadHunter Assay [10 μM ATP] - TW

Procedure Summary:

Human recombinant protein kinase PKCβ2 expressed in insect Sf21 cells is used. Test compound and/or vehicle is preincubated with 0.726 µg/ml enzyme in modified HEPES buffer pH 7.4 for 15 minutes at 37°C. The reaction is initiated by addition of 250 µg/ml histone type III-S, 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 type III-S formed. Compounds are screened at 10 µM.

Substrate:

250 µg/ml Histone

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