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Assay Method Information |
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| | HTRF Assay |
| Description: | An HTRF assay (Cisbio KinEASE-TK cat #62TKOPEC) was performed to quantitate the ability of test compounds to inhibit BTK mediated phosphorylation of substrate. Assays were assembled in 384 well plates where 6 nM of full-length human His-tagged BTK (Life Technologies cat #PV3587) and test compound at varying concentrations were preincubated for 15 minutes at 28 ° C. Then, 1 uM of TK substrate-biotin and 30 uM ATP were added and incubated for an additional 30 minutes at 28 ° C. Phospohrylation was detected by adding 62.5 nM Streptavidin-XL665 and TK-Antibody Cryptate diluted 1:100 in HTRF detection buffer (Cisbio cat #62SDBRDF) and incubated for 60 minutes at RT. The plate was read on an Envision plate reader and the fluoresence is measured at 620 nm (cryptate) and 665 nm (XL665). A ratio is calculated (665/620) and converted to POC relative to control and blank wells.Assay Buffer:50 mM HEPES (Invitrogen #15630), 0.01% Brij-35 (sigma #B4184), 10 mM MgC12 (Sigma M1028), 1 mM EGTA. |
| Affinity data for this assay | |
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