PI3Kalpha (PIK3CA) (p110a(E545K)/p85a) Mouse PtdIns(4,5)P 3-Kinase Enzymatic TR-FRET [Km ATP] KinaseProfiler LeadHunter Assay - FR

Procedure Summary:

PI3K p110a(E545K)/p85a (m) is incubated in assay buffer containing 10 µM phosphatidylinositol 4,5-bisphosphate and Mg/ATP (concentration as required). The reaction is initiated by the addition of the ATP solution. After incubation for 30 minutes at room temperature, the reaction is stopped by the addition of stop solution containing EDTA and biotinylated phosphatidylinositol- 3,4,5-trisphosphate. Finally, detection buffer is added, which contains europium-labelled anti-GST monoclonal antibody, GST-tagged GRP1 PH domain and streptavidin allophycocyanin. The plate is then read in timeresolved fluorescence mode and the homogeneous time-resolved fluorescence (HTRF) signal is determined according to the formula HTRF = 10000 x (Em665nm/Em620nm)

Substrate:

10 µM phosphatidylinositol 4,5-bisphosphate

ATP Concentration:

[ATP]= 45µM (Km= 48µM )

Incubation:

40 min at Room temperature

Control Inhibitor:

PI-103

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