| Assay Method Information | |
| | Back Scattering Interferometry Technology (BSI) |
| Description: | To measure the direct binding of small molecules with CFTR, the interaction between small molecules and membrane fragments derived from HEK293 cells over-expressing WT CFTR was investigated on the TruBind BSI System.HEK293 containing WT CFTR and HEK293 control membrane fractions were prepared as follows. HEK293 were transiently transfected with WT CFTR or left untreated, washed with PBS and collected in cold PBS supplemented with protease inhibitor cocktail (PIC). Cells were centrifuged and resuspended in homogenisation buffer (15 mM Tris-HCl pH 7.5, 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA+protease inhibitors). For compound testing HEK293 WT CFTR or HEK293 membrane fractions, at final concentration of 10 μg/mL in 50 mM Tris-HCl pH 7.5, 1 mM EDTA with 1.2% DMSO were mixed 1/1 with a serial dilution of the compound, starting from 10 μM, in 50 mM Tris-HCl pH 7.5, 1 mM EDTA with 1.2% DMSO. Mixtures were incubated at room temperature for 4 hours before being run on the BSI instrument. Samples were measured in quadruplicate in dual channel mode which allows the simultaneous measurement of specific, WT CFTR membranes (assay), as well as unspecific, control membranes (reference). For each assay the reference data is subtracted, point by point, from the assay data and plotted as fringe shift in units of radions. Each compound was run to have at least two successful experiments with good reproducibility. Success was defined as having a binding signal with a R2>0.7. The final data is exported to Graphpad Prism and fit with a one-site binding equation to determine a Kd for the assay. The data obtained using the method demonstrates clearly that the test compound as defined in the present claims directly bind to CFTR with low Kd. |
| Affinity data for this assay | |
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