| Assay Method Information | |
| | Surface Plasmon Resonance (SPR at 37 C) |
| Description: | Table B2: Biacore 8K buffer line was placed into 1 L of Immobilization buffer (20 mM HEPES pH 7.5/150 mM NaCl/5 mM MgCl2/0.5 mM TCEP/0.005% P20/500 nM GDP) and Change Solutions was performed. A Biacore Series S SA chip (Cytiva 29104992) was inserted and Change Solutions was performed 3 times. The chip surface was conditioned by injecting conditioning solution (40 mM NaOH/1 mM NaCl) in four pulses of 30 seconds at 30 uL/min. These pulses were followed by pulses of running buffer injected for 30 seconds at 30 uL/min. Normalization was run using the BiaNormalize solution (Cytiva 29207950). 300 nM Biotinylated KRas G12V 1-169 was injected over all active flow cells at a flow rate of 5 uL/min until reaching a response of 1000 RUs. The buffer line was switched into Running Buffer (20 mM HEPES pH 7.5/150 mM NaCl/5 mM MgCl2/0.5 mM TCEP/0.005% P20/500 nM GDP/2% DMSO) and Change Solutions was performed. Compounds were plated into a Greiner v-bottom 384-well Microplate (Greiner 781280) in 8-point dose-response with a log-fold dilution with a final volume of 2 uL in DMSO. 98 μL of non-DMSO containing buffer was backfilled into all compound wells (final volume of 100 uL with 2% DMSO). Solvent corrections of 1%, 1.5%, 2%, 2.5%, and 3% DMSO were plated into a Greiner U-bottom 96-well microplate (Greiner 650201). The SPR method was run with both flow cell and sample compartment at 37° C. with 5 startup cycles of 30 second association, and 30 second dissociation at a flow rate of 70 uL/min. An eight-point single cycle kinetics (SCK) analysis was run with 30 second association time, 1600 second dissociation time, 70 uL/min flow rate, 1600 sec stability period, and a 50% DMSO wash after injection. Solvent corrections were run at the end of the method. Results were analyzed with the Biacore Insights Evaluation Software using the Single Cycle Kinetics analysis method. Sensorgrams were fit using a 1:1 binding model. KD values, as an average of multiple runs, if applicable, are presented in Table B2 with two significant figures. |
| Affinity data for this assay | |
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