| Assay Method Information | |
| | HTRF based binding assay |
| Description: | An HTRF based binding assay was applied to assess the affinity of candidate compounds to Cbl-b protein. CBL-B (GST-Tag) was purchased from BPS (Cat #80415). A fluorescence labeled Cbl-b binding probe was made in-house. Anti GST-Tb cryptate monoclonal antibody was from Cisbio (61GSTTLB). In brief, compounds were diluted and transferred to opti-384F black plate (PE, 6007279) by echo. 1× Assay Buffer (HEPES pH 7.5 50 mM, NaCl 50 mM, MgCl2 5 mM, TCEP 1 mM, Tween-20 0.01%), 2× Protein mix (10 nM), and 2× Probe mix (120 nM) were prepared. 10 μL 2× Protein mix (or 1× Assay buffer, as no protein control) was added to assay plate. After 30 min incubation 25° C., 10 μl 2× Probe mix was added for another 2 h incubation at 25° C. Data were collected by Envision reading using Ex340/Em495/520. |
| Affinity data for this assay | |
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