| Assay Method Information | |
| | KRAS/SOS1 nucleotide exchange HTRF Assay |
| Description: | Reaction buffer was prepared with 10 mM HEPES 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.05% BSA, 0.0025% NP40, and 0.5% DMSO. Recombinant KRAS G12C protein (Recombinant human KRAS G12C mutant; wt Genbank accession# NM_033360.3; aa 2-169, expressed in E. coli with N-terminal GST fusion; MW 46 kDa) was preincubated with the anti-GST Tb antibody (Cisbio, category# 61GSTTLB) for 1 h at rt. SOS1 (Recombinant human SOS1; Genbank accession# NM_005633.3; aa 564-1049, expressed in E. Coli with C-terminal StrepII; MW=60.6 kDa) was prepared in reaction buffer. Compounds in 100% DMSO were added to assay wells with SOS1 reaction mixture (10 µL per well) using acoustic liquid dispensing (Echo 550 Series, Labcyte) and incubated for 15 minutes at rt. GTP-DY-647P1 was added to the GST-KRAS/anti-GST Tb antibody mixture and 5 uL of mixture was added to assay wells to a final assay volume of 15 µL. This mixture consisted of recombinant KRAS G12C (30 nM final concentration), recombinant SOS1 (20 nM final concentration) and GTP-DY-647P1 (150 nM final concentration). The reaction was monitored for 40 minutes at rt using Envision plate reader (Perkin Elmer; Ex/Em = (320-75/665-7.5; 615-8.5). Data analysis was performed when the reaction was linear, corresponding with the HTRF signal at approximately 20 min after addition of GTP-DY-647P1 and GST-Kras/anti-GST Tb antibody mixture. The background (wells with buffer only; no SOS1 protein added) subtracted signals were converted to % activity relative to DMSO controls. Data was analyzed using GraphPad Prism 4 with sigmoidal dose-response (variable slope); 4 parameters with Hill Slope. |
| Affinity data for this assay | |
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