PI3Kbeta (PIK3CB) (p110b/p85a) Human PtdIns(4,5)P 3-Kinase Enzymatic TR-FRET [10 uM ATP] KinaseProfiler LeadHunter Assay - FR

Procedure Summary:

PI3K (p110b/p85a) (h) 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 Mg/ATP mix. 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]= 10µM (Km= 235µM )

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