| Assay Method Information | |
| | RAS:RAF-RBD Binding Assay |
| Description: | The RAF-Ras binding domain (RBD) protein interaction assay utilizes recombinant biotinylated KRAS protein containing a G12D mutation (SEQ ID NO: 1) and the GST-tagged Ras binding domain of C-RAF (residues 50-132) from Jena Biosciences (catalog #PR-366). Compounds are added to KRAS and then after a 30-minute incubation time the RAF-RBD and detection antibodies are added. Small molecule inhibitors that block the interaction of c-RAF-RBD prevent generation of a TR-FRET signal. 2* Biotinylated KRAS G12D protein is diluted to 20 nM in assay buffer (20 mM HEPES pH 7.5, 150 mM sodium chloride, 10 mM magnesium chloride, and 0.01% Tween20). Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series in a 384-well low dead volume microplate (Labcyte, catalog #LP-0200). Once titrations are made, 50 nL of the diluted compounds is acoustically dispensed into 384-well plates (Corning, catalog #3820) using an Echo 655 (Labcyte). Each well of the assay plate receives 5 uL of Biotinylated KRAS G12D assay solution and is incubated at room temperature for 30 minutes. Each well then receives 5 uL of 100 nM GST-C-RAF RBD protein and a 1:100 dilution of both anti-GST-d2 (Cisbio catalog #61GSTDLA) and Streptavidin-Tb cryptate (Cisbio catalog #610SATLA) in assay buffer and the plate is mixed and briefly centrifuged followed by a 60-minute incubation at room temperature. The time-resolved fluorescence resonance energy transfer signal is measured on an Envision (PerkinElmer) plate reader: dichroic mirror=LANCE/DELFIA DUAL/Bias; Emissionl=615 nm; Emission2=665 nm; delay time=60 ms. The signal of each well is determined as the ratio of the emission at 665 nm to that at 615 nm. Percent effect of each well is determined after normalization to control wells containing DMSO (no effect) or a saturating concentration of inhibitor (max effect). The apparent effect as a function of compound concentration is fit to a four-parameter logistic equation. |
| Affinity data for this assay | |
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