| Assay Method Information | |
| | Biochemical Assay 2 |
| Description: | Preparation of GST-tagged hK-RasGl2C loaded with fluorescent nucleotide was performed as follows: incubation of 11.5 μM hK-RasGl2C with 5-fold excess GDP-Dy647 nucleotide (54 μM) in 500 μl NLS-buffer (RAS activation Kit Jena Bioscience, Kat. #PR-950) for 10 min at 37° C. Addition of 20 μl 1 M MgCl2 (Sigma) to final 40 mM and store on ice. Purification into buffer (10 mM HEPES pH 7.4 (Applichem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma)) by use of a PD-Minitrap desalting column (GE Healthcare). Concentration of 1 ml purified hK-Ras-GDP-Dy647 is approx. 4-5 μM.The assay buffer contained 10 mM HEPES pH 7.4 (Applichem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermofisher), 0.05% BSA Fraction V, pH 7.0, (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma). |
| Affinity data for this assay | |
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