| Assay Method Information | |
| | HPK1-SLP76 TR-FRET ASSAY |
| Description: | To each well of black Corning #3820384-well plate, an ECHO was used to dispense 7.5 nL of DMSO or Test compound in DMSO. A 1.5x kinase solution, 5 μL/well, was added and preincubated for 30 minutes before 2.5 μL/well of 3x substrate solution was added. The reaction solution incubated for 60 minutes and quenched with 2.5 μL of 4x detection solution. The solutions were incubated for an additional 60 minutes prior to reading on a Perkin Elmer Envision. The TR-FRET signal was measured at both 615 and 665 nm. The calculated emission ratio of 665/615 was used to determine the percent effect for each compound concentration. Detailed HPK1 Assay Protocol:Compounds are serially diluted (3-fold in 100% DMSO) across a 384-well polypropylene source plated from column 3 to column 12 and column 13 to column 22, to yield 10 concentration dose responses for each test compound. Columns 1, 2, 23 and 24 contain either only DMSO or a pharmacological known control inhibitor. Once titrations are made, 7.5 nL of the compounds on 384 well plates are transferred by acoustic dispersion into a 384-well assay plate (Corning 3820) to assay the HPK1 enzyme.The HPK1 kinase biochemical assay was developed using commercially available HTRF reagents. The assay contains the following reagents: 1) Assay Buffer: 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij−35, 0.05 % BSA and 0.5 mM TCEP; 2) Enzyme Solution: HPK1 (Carna); 3) Substrate Solution: ATP and Full Length SLP76 with His-Tag; 4) Stop and Detection Solution: EDTA, LANCE Eu-W1024 Anti-6xHis Ab (Perkin Elmer) and Phospho-SLP-76 (Ser376) (D7S1K) XP Rabbit mAb (AF 647 Conjugate) (Cell Signaling Technologies). Enzyme, Substrate and Stop/Detection solutions are prepared in assay buffer. Enzyme solution (75pM HPK1 Final), 5μL/well, is added to 384-well assay plate and incubated with 7.5nL of compound or DMSO for 30 minutes. Kinase reaction is initiated with addition of 2.5 μL of substrate solution (ATP 10uM and SLP7610 nM Final) and allowed to proceed for 60 minutes. Enzyme addition and compound pre-incubation are initiated by the addition of 5 μL of HPK1 enzyme solution (at one and a half times its final concentration of 75pM) to all wells using a BioRaptr. Plates are incubated at room temperature for 30 minutes. Reactions are initiated by addition of 2.5 μL of 3x substrate solution (10 nM SLP76 and 10 uM ATP FInal) using BioRaptr. Plates are incubated at room temperature for one hour. Reactions are quenched, and activity detected by addition of 2.5 μL of 4x stop and detection solution (10 mM EDTA, 0.75 nM LANCE Eu-W1024 Anti-6xHis Ab and 0.75 nM Phospho-SLP-76 (Ser376) (D7S1K) XP Rabbit mAb (AF 647 Conjugate) Final) to all wells using the BioRaptr. Following a one-hour incubation, the HTRF signal is measured on the Envision plate reader set for 320nm excitation and dual emission detection at 615nM (Eu) and 665nM (AF647). |
| Affinity data for this assay | |
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