Hepes free acid

E. coli BL21 cells were obtained from New England Biolabs (Ipswich, MA), and Bacto yeast extract, agar and tryptone for bacterial growth were from Gibco (Waltham, MA). Ampicillin and HEPES free acid were from Research Products International (Mount Prospect, IL). Kanamycin and reduced L-glutathione were from Sigma-Aldrich (St. Louis, MO). Phenylmethylsulfonyl fluoride (PMSF) used to inhibit proteolysis during Ras purification was from Roche Diagnostics (Basel, Switzerland). TALON cobalt(II) metal affinity resin for polyhistidine tag purification was from Takara (Kusatsu Japan), and HYDRANAL imidazole was from Honeywell (Charlotte, NC). Ethylenediamine tetracetic acid disodium salt dihydrate (EDTA) was from Thermo Fisher Scientific (Waltham, MA). Non-hydrolyzable GTP analog GMPPNP, conjugated with four lithium counter ions, at >95% purity, was from Abcam (Cambridge, UK). Guanosine 5’-diphosphate [GDP] disodium salt was from Sigma-Aldrich (St. Louis, MO). Desalting columns were Econo-Pac 10DG desalting prepacked gravity flow columns from Bio-Rad (Hercules, CA). Vivaspin 500, 10,000 MWCO spin concentrators from Sartorius were used to concentrate protein samples (Göttingen, Germany). Microscale thermophoresis measurements were performed on a Nanotemper Monolith NT.115 instrument utilizing Monolith NT.115 capillaries (Munich, Germany).