| Assay Method Information | |
| | Fluorescence Polarization (FP) VHL Binding Assay |
| Description: | The binding of test compounds to the VHL Elongin B/C complex was measured using a fluorescence polarization tracer competition assay. The VHL / Elongin B/C protein complex used in the assay was 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 was co-expressed with Elongin B (residues M1-Q118) and Elongin C (ResiduesM17-C112) in E. coli. The VHL / Elongin B/C complex was 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 I Elongin B/C complex was dialyzed into formulation buffer: 20mM Bis-Tris pH7.0, 150mM NaCl, ImM DTT. A VHL fluorescence polarization probe consisted of a VHL ligand coupled to carboxytetramethylrhodamine (TAMRA); (2S,4R)-N-(2-(2-(3',6'-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-5-carboxamido)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-l-((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide. Compounds were 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 was dispensed into wells designated for “VHL control” (without compound) wells. The “Assay Buffer” consisted 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 pM VHL Elongin B/C complex was prepared and 5 l dispensed using a BioRapTR (Beckman Coulter) into each well of the assay plate. Assay Buffer was also dispensed into “no VHL control” wells using the same method. A “pre-assay” fluorescence measurement was 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 was prepared in Assay Buffer and 5pl dispensed into each well of the assay plate using a BioRapTR (Beckman Coulter). The final VHL I Elongin B/C protein concentration is 2.64 nM and the final probe concentration is 1.67 nM. Assay plates were briefly centrifuged and incubated for 1 hour at room temperature. “Post-assay” fluorescence polarization measurements were made as described for the “pre-assay” fluorescence measurement. Fluorescence polarization was 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 were analyzed using Genedata Screener software and normalized to the “no VHL control” and “VHL control” (without compound). IC50 values were calculated using a four parameter curve fit (Robust method). |
| Affinity data for this assay | |
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