5'-Atp → HCID 58423310, compound

Participants

Reaction Steps

One determines phosphorylation of the FL-Ahx-Bad in the presence and absence of the compound of interest. One preincubates 5u1 of Enzyme/Substrate/adenosine triphosphate (ATP) mix consisting of 2.4 nM PIM1, 3.6 uM FL-Ahx-Bad, and 240 uM ATP in 1.2× buffer with 2 ul of compound for 20 minutes at 25° C. One initiates reactions with 5 ul of Metal mix consisting of 24 mM MgCl2 in 1.2× buffer and incubated at 25° C. for 90 minutes and stops the reactions by addition of 5 ul of Stop mix consisting of 100 mM HEPES, 121 mM ethylenediamine tetraacetic acid, 0.8% Coatin Reagent 3 (Caliper, Mass.), and 0.01% Tween. One detects phosphorylated and unphosphorylated substrate by a Caliper LC3000 reader (Caliper, Mass.) in the presence of separation buffer consisting of 100 mM HEPES, 16 mM ethylenediamine tetraacetic acid, 0.1% Coatin Reagent 3 (Caliper, Mass.), 0.015% Brij-35, 5% DMSO, and 5.6 mM MgCl2. One may use the following separation conditions for a Caliper LC3000: −1.0 PSI, −2000 V upstream voltage, −400 V downstream voltage, 0.2 second sample sip, 45 second post sip, 10% laser strength.

Provenance & Evidence