| Assay Method Information | |
| | KRas G12D Surface Plasmon Resonance (SPR) Binding Assay |
| Description: | Briefly, 1L of 1.05X HBS-Mg buffer (262.5 mM Bioultra Hepes, pH 7.5, 157.5 mM NaCl, 105 mM MgCl2, 0.525 mM TCEP, 0.0305% Brij-35) was prepared and filter sterilized using a 0.22 m bottle top filter. Approximately 50 mL of 1.05X HBS-Mg buffer was removed and saved for future dilutions. A 50 mL aliquot of DMSO (Sigma Aldrich DMSO Lot. #SHBK2079) was added and continued to stir for 10 minutes, creating the final 1.0X HBS-Mg buffer (250 mM Bioultra Hepes pH 7.5, 150 mM NaCl, 100 mM MgCl2, 0.5 mM TCEP, 0.03% Brij-35). Biacore T200 instrument was primed using 1.0X HBS-Mg buffer before docking a GE Streptavidin (SA) chip and then primed two additional times prior to beginning the immobilization step. All immobilized protein mixtures were created using 3-5 mg/mL Biotinylated Avidin-tagged KRAS protein using the following immobilization settings: SA chip type, 1 flow cells per cycle, 720 second contact time, and 5 ul/min flow rate. Normalization of the detector was also performed during the immobilization step using the GE BiaNormalize solution. All compounds were diluted to 10 mM in 100% DMSO prior to being diluted 20X in 1.05X buffer. Another 10X dilution was created using 1.0X buffer prior to performing a series of 3X dilutions to create a compound concentration curve using the following assay settings: 20C analysis temperature, General Settings=10 Hz data collection rate and multi-detection; Assay Steps=all set to LMW kinetics; Cycle Types=LMW kinetics (60 s contact time, 120 s dissociation time, 100 ul/min flow rate, extra wash after injection with 50% DMSO, flow path 1,2,3,4); Flow path detection=2-1, 4-3). |
| Affinity data for this assay | |
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