ATR /ATRIP Human PIKK Kinase Enzymatic [Km ATP] KinaseProfiler LeadHunter Assay - FR

Procedure Summary:

ATR/ATRIP (h) is incubated in assay buffer containing 50 nM GST-cMyc-p53 and Mg/ATP (concentration as required). The reaction is initiated by the addition of the Mg/ATP mix. After incubation for 30 minutes at room temperature, the reaction is stopped by the addition of stop solution containing EDTA. Finally, detection buffer is added, which contains d2-labelled anti-GST monoclonal antibody, and a Europium-labelled anti-phospho Ser15 antibody against phosphorylated p53. The plate is then read in time-resolved fluorescence mode and the homogeneous time-resolved fluorescence (HTRF) signal is determined according to the formula HTRF = 10000 x (Em665nm/Em620nm).

Substrate:

50 nM GST-cMyc-p53

ATP Concentration:

[ATP]= 10µM (Km= 0.17µM )

Incubation:

40 min at Room temperature

Control Inhibitor:

VE-822

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