| Assay Method Information | |
| | TR-FRET Biochemical Assay |
| Description: | The KRAS: CRAF PPI assay can be utilized to identify KRAS ‘ON’ state inhibitors, as the KRAS protein is loaded with GMPPNP (a non-hydrolyzable GTP analog), which represents the active KRAS ‘ON’ state. FLAG-tagged CRAF RBD binding to the GMPPNP loaded biotin-tagged KRAS will result in a fluorescence energy transfer from the donor (Tb-anti-FLAG, emission 620 nm) to the acceptor (SA-XL665, emission 665 nm). Disruption of KRAS and CRAF binding will reduce the TR-FRET signal. Test compound was dissolved in DMSO to create a 1 mM stock solution. A 45 μL volume of stock solution was transferred to a 384well polypropylene plate. A 3fold, 10point dilution was performed by transferring 15 μL of test compound solution into 30 μL of DMSO. Assay buffer A was prepared with 25 nM Hepes, pH7.3, 0.002% Tween 20, 0.1% BSA, 100 nM NaCl, 5 mM MgCl2, and 10 uM GMP-PNP. 4× KRAS Solution was prepared with 80 nM of Biotin-KRAS (G12D/V), GMPPNP loaded or Biotin-KRAS (WT), GMPPNP loaded protein in assay buffer A. 4× Raf RBD Solution was prepared with 400 nM FLAG CRAF RBD in assay buffer A. 2× Detection Mixture was prepared with 2 nM Tb-anti-FLAG antibody and 10 nM and 10 nM SA-XL665 in assay buffer A. A volume of 100 nL of 100× test compound working solution or DMSO vehicle control solution was dispensed to each well (duplicate for each concentration) of a 384-well ProxiPlate Plus assay plate. A volume of 2.5 μL of 4× KRAS Solution was added to each well of the assay plate for a final concentration of 20 nM Biotin-KRAS (G12D/V),GMPPNP loaded or 20 nM Biotin-KRAS (WT), GMPPNP. For low control, add 2.5 μL of assay buffer (no KRAS protein). Pre-incubate assay plate for 30 minutes at room temperature. Following pre-incubation of test compound or DMSO with Biotin-KRAS (G12D/V),GMPPNP loaded or Biotin-KRAS (WT),GMPPNP, a volume of 2.5 μL of 4× FLAG cRAF RBD Solution was added to each well of the assay plate for a final concentration of 100 nM FLAG CRAF RBD. The assay plate was subjected to centrifuge at 1000 rpm for 1 minute and pre-incubate assay plate for 150 minutes at room temperature. Following the pre-incubation with FLAG cRAF RBD, a volume of 5 μl of 2× Detection Mixture was added to each well of the assay plate for a final concentration of 1 nM Tb-anti-FLAG and 5 nM SA-XL665. The assay plate was centrifuged at 1000 rpm for 1 minute and incubated for 90 minutes at room temperature. TR-FRET signal was measured on a BioTek Synergy Neo2 microplate reader (excitation at 340 nm, emission at 665 nn and 620 nm). |
| Affinity data for this assay | |
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