| Assay Method Information | |
| | TR-FRET Assay |
| Description: | 1) KRAS nucleotide exchange buffer20 mL of 1000 mM HEPES, 20 mL of 500 mM EDTA, 10 mL of 5 M sodium chloride, 0.1 mL of 100% Tween 20, and 949.9 mL of water were weighed and formulated to 1 L of solution. The solution was sterilized by filtration and stored at 4° C.2) KRAS assay buffer20 mL of 1000 mM HEPES, 10 mL of 1000 mM magnesium chloride, 30 mL of 5 M sodium chloride, 0.05 mL of 100% Tween 20, and 939.95 mL of water were weighed and formulated to 1 L of solution. The solution was sterilized by filtration and stored at 4° C.3) KRAS/Bodipy GDP/Tb-SA mixture9.5 μL of 95 μM KRASG12D protein and 440.5 μL of KRAS nucleotide exchange buffer were weighed and mixed. The mixture was incubated at room temperature for 1 hour and formulated to 1 L of solution together with 8.4 μL of 17.9 μM Tb-SA, 1.8 μL of 5 mMBodipy GDP and 9539.8 μL of KRAS assay buffer. After mixing, the solution was left to stand at room temperature for 6 hours, and stored at −80° C.b. Assay reagents:1) KRAS enzyme solution73.3 μL of KRAS/Bodipy GDP/Tb-SA mixture and 2126.7 μL of KRAS assay buffer were weighed and formulated to 2200 μL of solution.2) SOS/GTP mixture1.59 μL of 166 μM SOS protein, 198 μL of 100 mM GTP and 2000.41 μL of KRAS assay buffer were weighed and formulated to 2200 μL of solution.4. Assay process1) The concentration of a stock solution of the control compound was 1 mM, and the concentration of a stock solution of compounds to be assayed was 10 mM. 9 μL of the control compound and compounds to be assayed were transfered to a 384-LDV plate;2) The compounds on the LDV plate were serially diluted 3-fold with Bravo to 10 concentrations;3) 9 nL of the compounds on the LDV plate were transfered to an assay plate using ECHO;4) 3 μL of 3 nM Kras/0.5 nM TB-SA/30 nM BodipyGDP mixture and 3 μL of Ras buffer were sequentially added to each well of the assay plate using a Dragonfly autosampler, and the assay plate was centrifuged at 1000 rpm/min for 1 minute;5) The assay plate was incubated at room temperature for 1 hour;6) 3 μL of 120 nM SOS/9 mM GTP mixture was added to each well of the assay plate using a Dragonfly autosampler, and the assay plate was centrifuged at 1000 rpm/min for 1 minute;7) The assay plate was incubated at room temperature for 1 hour;8) The plate was read with Envision and data were recorded;9) The data were analyzed using Excel and Xlfit, and ICso of the compounds to be assayed were calculated. |
| Affinity data for this assay | |
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