Ni nta resin

Mutants of mEos2 in the pRSETa vector (Invitrogen) were made by QuikChange (Agilent) or Kunkel ^{30 (link)} mutagenesis in E. coli XL1-Blue (Agilent). Point mutants were created first, with subsequent combination of beneficial mutations. Proteins were produced at 37 °C in E. coli BL21(DE3) grown in Studier auto-induction media ^{31 (link)} supplemented with 60 μg/ml ampicillin. Bacterial pellets were then re-suspended in 30 ml “native lysis” buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0, adjusted with NaOH; protease inhibitors). Cell pellets were mechanically disrupted with an EmulsiFlex-C5 high-pressure homogenizer (Avestin). The supernatant was incubated overnight with 50% Ni-NTA resin (Bio-Rad) by rotating slowly on a rotary shaker, at 4 °C. The pre-equilibrated solution was carefully loaded onto an empty, clean 20 ml column (1.5 × 12 cm polypropylene; Bio-Rad), and purified by gravity-flow chromatography (batch purification). The flow-through was collected by washing 4 times with 20 ml “native wash” buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0, adjusted with NaOH) and kept for SDS-PAGE analysis, followed by five to six 2 ml fractions collected in “elution” buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0, adjusted with NaOH) and kept for SDS-PAGE analysis.

0.5 ml of Ni-NTA resin (1 ml of slurry; Qiagen) was first washed with water and then equilibrated with wash buffer (300 mM NaCl, 10% glycerol, 20 mM Tris-HCl pH 8, 20 mM imidazole, 0.06% DDM, 0.02% CHS, 0.5 mM TCEP). The ~40 ml of solubilized membrane suspension was incubated for 2 hours with the pre-equilibrated Ni-NTA resin, at 4°C on a rotating platform. Then, the Ni-NTA resin was loaded onto a 1 ml glass column (BioRad) while Abs₂₈₀ and buffer conductivity were monitored with a low-pressure chromatography system (Biologic-LP, BioRad). Wash buffer was passed through the resin at 0.5 ml/min until Abs₂₈₀ returned to baseline value (~20 column volume). Protein was eluted with elution buffer containing 300 mM imidazole, 300 mM NaCl, 10% glycerol, 20 mM Tris-HCl pH 7.5, 0.06% DDM, 0.02% CHS, 0.5 mM TCEP) and 1 ml fractions were collected manually. Aliquots of each fraction (15 μl) were analyzed by 10% SDS-PAGE and the best fractions were pooled for the His-tag removal step.

DNA encoding K, N, H-Ras fused with N-terminal multi-histidine tags (or GST in the case of H-Ras) were cloned into pJExpress vectors (DNA 2.0, Menlo Park, CA). R-Ras and Rap1B were cloned into modified pET28a vectors with N-terminal histidine tags. Rac1, RhoA, and CDC42 were cloned into pET24a vectors with C-terminal histidine tags. All proteins were overexpressed in the E coli. B21 strain. Cultures for each were grown in Luria broth at 37 °C with shaking at 225 rpm until the OD600 reached 0.8. The temperature was reduced to 16 °C and expression was induced with 0.5 mM IPTG. After 8–12 hours, bacteria were harvested by centrifugation for 15 minutes at 4 °C. The pellet was resuspended in lysis buffer (20 mM NaH₂PO₄, 500 mM NaCl, 5% glycerol, 20 mM imidazole) containing 1 mM PMSF, 1 mM benzamidine, and 1 mg/ml lysozyme then flash frozen. Cells were lysed to completion using sonication and the lysates clarified by centrifugation for 35 minutes. Supernatants were filtered using 0.22 μm syringe filters, and applied to Ni-NTA resin (Biorad, Hercules, CA). Elution was accomplished with a step gradient to buffer containing 20 mM NaH₂PO₄, 1 M NaCl, 5 % glycerol, and 100 mM imidazole. Proteins were immediately buffer exchanged using a desalting column into Buffer A (20 mM Tris-HCl, 100 mM NaCl, pH 8.0).