| Assay Method Information | |
| | Cbl-b Biochemical Assay (TR-FRET) |
| Description: | Fluorescein-BODIPY labeled UBED2D(C85K)-Ub was prepared by conjugating ubiquitin (Ub) labeled at its N-terminus with Fluorescein-BODIPY maleimide (ThermoFisher Catalog no B10250) to E. coli expressed and purified UBED2D(C85K) [see Dou et al Nature Structural and Molecular Biology 8: 982-987, 2013]. Recombinant human UBE2D2(C85K) was expressed in E. coli, purified and ubiquitinated and Bodipy labelled in vitro. Protein was diluted to 200 nM in assay buffer without MgCl2 (or Cisbio PPI buffer). Streptavidin-Terbium was added to 2 nM and EDTA to 10 mM, to provide a binding assay mix.Compounds were dissolved in DMSO and diluted to prepare a ten-point dilution series. 100 nl of each compound concentration was dispensed in duplicate in a 384 well black assay plate using acoustic dispensing. Wells for maximum signal controls received 100 nl of DMSO only and wells for minimum signal controls received 100 nl of a reference inhibitor compound at a final assay concentration of 100 mM to produce 100% inhibition.5 μl of diluted Cbl-b enzyme was added to all wells of the assay plate and incubated at RT for 30-60 min. The enzyme assay was initiated by addition of 5 μl of Src-Zap/ATP mix to all wells, and the plate incubated at RT for 60 min. The enzyme reaction was terminated and the binding reaction was initiated by adding 10 μl of binding assay mix to all wells and incubating the plate at RT for 60 min prior to assay read.Final assay conditions consisted of 6 nM Cbl-b, 1-2.5 nM Src-Zap70, 0.5 mM ATP, 1% (v/v) DMSO (enzyme reaction) and 100 nM UBE2D2(C85K)-Ub-FL-BODIPY, 5 mM EDTA, 1 nM Streptavidin-Tb (binding reaction).The HTRF assay signal was measured at 520 nm on an Envision plate reader, with reference signal at 485 or 620 nm. Data was normalized using maximum and minimum assay controls: % Inhibition=100−(100*((maximum control)−unknown)/(maximum control−minimum control)). A 4-parameter dose-response equation was used to fit the normalized dose-response data and derive an IC50 for test compounds. |
| Affinity data for this assay | |
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