mTOR / FKBP12 Human PIKK Kinase Enzymatic Radiometric [Km ATP] KinaseProfiler LeadHunter Assay - FR

Procedure Summary:

mTOR/FKBP12 (h) is incubated with 50 mM HEPES pH 7.5, 1 mM EGTA, 0.01% Tween 20, 2 mg/mL mTOR substrate, 10 µM FKBP12, 3 mM MnCl2, and [gamma-33P]-ATP (specific activity and concentration as required). The reaction is initiated by the addition of the Mn/ATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of phosphoric acid to a concentration of 0.5%. An aliquot of the reaction is then spotted onto a filter and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting.

Substrate:

2 mg/mL mTOR substrate

Tracer:

33P

ATP Concentration:

[ATP]= 45µM (Km= 35.5µM )

Incubation:

40 min at Room temperature

Control Inhibitor:

Rapamycin

Test Sample Requirements:

Please provide compounds dissolved in 100% DMSO (PBS or HBSS prohibited).
Stock solution should be ≥50× the final assay concentration (a 10 mM stock is also acceptable).
Required volume:
– <60 kinase assays: 120 µL of 50× stock
– ≥60 assays: 420 µL of 50× stock
If you supply 10 mM stocks or powder (pre-weighed), we will apply the appropriate conversion.
For batches of ~50 compounds supplied as solids, the TAT will be extended.

Minimum Order Quantity:

2