| Assay Method Information | |
| | BTK Target Occupancy |
| Description: | Ramos B cells (ATCC, cat no. CRL-1923) were plated in 24-wells culture plates at 2×106 cells per well in a total volume of 900 μL DMEMF12+10% FBS+2 mM L-Glutamine+Pen/Strep. Allow the cells to rest 1 h at 5-7% CO2 and 37° C.Serial dilutions log 10 from 10 mM to 316 nM of test compounds are made in 100% DMSO, followed by a 100-fold dilution into culture medium.For each well, 100 μL was then transferred to well plate containing 900 μL of Ramos B cells. Final compound concentration range in the assay varied from 10 μM to 0.316 nM, with a final DMSO concentration of 0.1% and incubated at 5-7% CO2 and 37° C. for 2h. Afterwards, cells are collected for the measurement of the BTK target occupancy using the BTK target occupancy ELISA as outlined below.The percent of drug-bound BTK in Ramos B cell samples was determined by an ELISA based method as follows: OptiPlate 96-well plates (Perkin Elmer) were coated with 125 ng/well anti-BTK Ab (BD Biosciences) and blocked with BSA (Sigma-Aldrich). Samples containing Ramos B cells were lysed in ice cold lysis buffer containing 50 mM Tris-HCl pH 7.5, 250 mM sucrose, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 0.05% digitonin, and protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were then incubated for 1 h in the absence or presence of 1 μM acalabrutinib, a saturating concentration that results in complete BTK occupancy. Final amount of cell lysate used per well in BTK target occupancy ELISA is representative of 2×105 Ramos B cells. The difference with the signal of the cell lysates not incubated with an excess acalabrutinib represents free BTK (not occupied by a BTK inhibitor). Samples were incubated for 1 h with biotin tag compound of Formula (II) (100 nM). This probe will bind covalently to Cys481 in the ATP pocket in BTK when the ATP pocket is not occupied by a covalent BTK inhibitor. Each sample was then added in duplicate to the prepared Optiplate and incubated for 2h at ambient temperature. Plates were washed with PBS+0.05% Tween20 four times. Streptavidin-HRP (Invitrogen; ELISA grade) was added at 100 μL/well (120 ng/mL) and incubated for 1 hour at room temperature. Plates were washed with PBS+0.05% Tween20 three times and then washed with PBS (without Tween 20) two times. One hundred L/well of SuperSignal ELISA Femto Substrate (ThermoFisher Scientific) was added and then chemiluminescence was measured after 1 minute (EnVision plate reader; PerkinElmer). |
| Affinity data for this assay | |
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