| Assay Method Information | |
| | Cbl-b Activity Assay |
| Description: | Experiment purpose: Testing of inhibitory activity of compounds on the interaction between Cbl-b protein and UbcH5B-Ub. Experimental method: Eu-Ubquitin-UbcH5B was prepared by incubating Eu-Ubquitin (Cisbio) with UbcH5B (ENZO), E1 (ENZO) at 37° C. for 4 hours. Eu-Ubquitin-UbcH5B was aliquoted and stored at −80° C. Cbl-b activity assay was performed in 384-well plates (Perkin Elmer). 100 nL of 3-fold serial dilutions of compounds (final concentration 10 μM-0.5 nM, starting concentration 10 μM, 3-fold dilution, 10 points, 10th point being 0.5 nM) and 5 μL of 50 nM Biotin-Cbl-b protein (Sigma) were incubated for 1 hour at room temperature, and the reaction buffer was 50 mM HEPES pH 7.0 (Gibco), 100 mM NaCl (Sigma), 0.01% Triton X-100 (Sigma), 0.01% BSA (Sigma) and 1 mM DTT (Invitrogen). 5 μL of Src mixed solution (40 nM Src (R&D), 2 mM ATP (Sigma), 10 mM MgCl2 (Sigma)) was added to the reaction plate and incubated at room temperature for 3 hours. 10 μL of detection solution (12.5 nM Strepdividin-XL665 (Cisbio), 500 nM Eu-Ubquitin-UbcH5B, 120 nM EDTA (Invitrogen), 0.004% BSA (Sigma)) was added to the reaction plate, incubated overnight at room temperature, and the HTRF signal (665 nm/615 nm) was read on Envision (Perkin Elmer). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |