| Assay Method Information | |
| | Fluorescence Polarization (FP) VHL Binding Assay |
| Description: | The binding of test compounds to the VHL Elongin B/C complex is measured using a fluorescence polarization tracer competition assay. The VHL / Elongin B/C protein complex used in the assay is generated as follows. The coding region for amino acids E55-D213 of human VHL with N-terminal His6 tag with a TEV protease cleavage site is co-expressed with Elongin B (residues M1-Q118) and Elongin C (ResiduesM17-C112) in E. coli. The VHL / Elongin B/C complex is purified using an affinity nickel column, anion exchange HiTrap QP HP column chromatography, and gel filtration using a Superdex 7526/60 column. The purified VHL / Elongin B/C complex is dialyzed into formulation buffer: 20mM Bis-Tris pH7.0, 150mM NaCl, 1mM DTT. A VHL fluorescence polarization probe consists of a VHL ligand coupled to carboxytetramethylrhodamine (TAMRA); (2S,4R)-N-(2-(2-(3',6'-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamido)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide. Compounds are prepared as a serial dilution in DMSO at a concentration 25-fold higher than the final desired concentration and acoustically dispensed (400 nl) into a ProxiPlate-384 Plus F, Black 384-shallow well Microplate (Part Number 6008260). DMSO is dispensed into wells designated for VHL control (without compound) wells. The Assay Buffer consists of 50 mM Tris pH 8.0, 120 mM NaCl, 0.005% Nonidet P-40, and 1% DMSO (v/v). Assay Buffer containing 5.28 μM VHL Elongin B/C complex is prepared and 5μl dispensed using a BioRapTR (Beckman Coulter) into each well of the assay plate. Assay Buffer is also dispensed into no VHL control wells using the same method. A pre-assay fluorescence measurement is made using an Infinite M1000 (Tecan) plate reader (Excitation 530 nm, Emission 574 nm, Bandwidth 10 nm). Assay Buffer containing 3.34 nM of the VHL FP probe is prepared in Assay Buffer and 5μl dispensed into each well of the assay plate using a BioRapTR (Beckman Coulter). The final VHL / Elongin B/C protein concentration is 2.64 nM and the final probe concentration is 1.67 nM. Assay plates are briefly centrifuged and incubated for 1 hour at room temperature. Post-assay fluorescence polarization measurements are made as described for the pre-assay fluorescence measurement. Fluorescence polarization is calculated for each sample; taking into account the pre-assay fluorescence measurements and subtracting the fluorescence signal of the compound/VHL only ( pre-assay ) measurements from the post-assay fluorescence polarization measurements, for each plane of polarization. The data are analyzed using Genedata Screener software and normalized to the no VHL control and VHL control (without compound). IC50 values are calculated using a four parameter curve fit (Robust method). |
| Affinity data for this assay | |
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