| Assay Method Information | |
| | Alpha Screen Assay |
| Description: | Purified full-length inactive BTK (wild type and C481 mutant, N-terminal 6XHIS tagged BTK, Mwt=78.2 kDa) were activated using soluble inositol hexakisphosphate (IP6) and ATP as described (Q. Wang, E. M. Vogan, L. M. Nocka, C. E. Rosen, J. A. Zorn, S. C. Harrison J. Kuriyan, eLife 2015; 4:e06074), with minor modification. To 190 μl of 1 mM IP6 in activation buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5% glycerol) was added 10 μl of inactive BTK at 5-6 mg/ml and incubated for 10 min at room temperature followed by addition of 200 μl of 2 mM ATP in assay buffer (50 mM Tris, pH 8, 10 mM MgCl2, 1 mM EDTA, 0.1 mM NaF, 0.02 mg/ml BSA, 10% glycerol, 2 mM sodium orthovanadate, 0.25 mM DTT) for a further 10 min. The activated BTK was frozen in 50 μl aliquots (125-150 μg/ml).BTK activity was assayed using a PLCγ2-derived biotinylated peptide substrate (biotin-EELNNQLFLYDTHQNLR-OH) and AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay) technology. The extent of peptide phosphorylation was determined by using acceptor beads conjugated to phosphotyrosine antibody that recognized the phosphorylated peptide and donor beads conjugated to streptavidin that binds to the biotin on the peptide. Excitation of the donor beads converted ambient oxygen to excited singlet oxygen which when in close proximity to acceptor beads, reacted with acceptor beads resulting in signal amplification.Test inhibitors and controls were prepared in 10% DMSO at 10-fold the desired final concentration and added to each well of a reaction plate (Corning 96-well half-area solid white nonbinding surface plate) in a volume of 2.5 μl. Full-length activated BTK (wildtype or mutant C481S) diluted to 0.179 nM in assay buffer (50 mM Tris, pH 8, 10 mM MgCl2, 1 mM EDTA, 0.1 mM NaF, 0.02 mg/ml BSA, 10% glycerol, 0.2 mM Na3VO4, 0.1 mM beta-glycerophosphate, 0.25 mM DTT) was added to each well in a volume of 17.5 μl and incubated with the inhibitors for 30 min. The kinase reaction was initiated by the addition of 5 μl of biotin-PLCγ2 peptide and ATP mixture diluted in assay buffer for a final concentration in the 25 μl reaction of 150 nM and 180 μM respectively and 0.125 nM enzyme. The plate was incubated for 120 min at room temperature and the reaction stopped by the addition of 10 μl stop/detection mixture containing 35 mM EDTA, 50 ug/ml AlphaScreen Streptavidin Donor beads (final concentration is 500 ng/well) and AlphaScreen Phospho-tyrosine (P-Tyr-100) Acceptor beads (final concentration is 500 ng/well) under green light conditions. The plate was incubated overnight at room temperature in the dark and the plates read on the BMG PolarStar Omega (excitation wavelength: 640 nm, emission wavelength: 570 nm, Gain=4000). Data were archived and analyzed with a 4-parameter fit to generate IC50 values using the CDD Vault from Collaborative Drug Discovery. |
| Affinity data for this assay | |
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