N-Terminal strep-6xHis-TEV mTagBFP2 RanQ69L was cloned into the pST50 vector via Gibson Assembly (New England Biolabs). The plasmid was transformed into E. coli Rosetta2 cells (EMD Millipore) for protein expression. Cells (4 L) were grown until OD600 = 0.8 at 37 °C in LB media, and then protein expression was induced using 500 mM IPTG for 18 hours at 16 °C before cells were pelleted. RanQ69L was purified in the following manner:24 (link),25 (link) we lysed cells by using an Emulsiflex french press (Avestin) in binding buffer (100 mM Tris–HCl, 450 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 2.5 mM PMSF, 6 mM BME, pH 8.75). We centrifuged the lysate at 20 000 × g and loaded the supernatant onto a StrepTrap HP column (GE Healthcare, 5 ml). Protein was eluted in binding buffer with 2.5 mM d -desthiobiotin, and dialyzed overnight into CSF-XB buffer (10 mM HEPES, 100 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10% sucrose w/v, pH 7.7). We included 200 μM GTP in lysis and elution buffers and obtained about 0.5 ml of 200 μM RanQ69L.
Emulsiflex french press
Manufactured by Avestin
The Emulsiflex french press is a laboratory equipment designed for high-pressure homogenization. It utilizes a hydraulic ram to apply high pressure on a sample, resulting in the disruption of cell membranes and the creation of a fine emulsion or suspension.
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2 protocols using emulsiflex french press
1
Purification of Mutant Ran GTPase
2
Purification of RanQ69L Protein
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N-terminal strep-6xHis-TEV mTagBFP2 RanQ69L was cloned into the pST50 vector via Gibson Assembly (New England Biolabs). The plasmid was transformed into E. coli Rosetta2 cells (EMD Millipore) for protein expression. Cells were grown until OD600= 0.8 at 37 °C in LB media, and then protein expression was induced using 500 mM IPTG for 18 hours at 16 °C before cells were pelleted.
RanQ69L was purified mostly as described (21, 22) . Briefly, cells were lysed using an Emulsiflex french press (Avestin) in binding buffer (100 mM Tris-HCl, 450 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 2.5 mM PMSF, 6 mM BME, pH 8.75). The lysate was centrifuged at 20,000 x g and the supernatant was loaded onto a StrepTrap HP column (GE Healthcare). Protein was eluted in binding buffer with 2.5 mM D-desthiobiotin, and dialyzed overnight into CSF-XB buffer (10 mM HEPES, 100 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10% sucrose w/v, pH 7.7). 200 μM GTP was included in lysis and elution buffers.
RanQ69L was purified mostly as described (21, 22) . Briefly, cells were lysed using an Emulsiflex french press (Avestin) in binding buffer (100 mM Tris-HCl, 450 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 2.5 mM PMSF, 6 mM BME, pH 8.75). The lysate was centrifuged at 20,000 x g and the supernatant was loaded onto a StrepTrap HP column (GE Healthcare). Protein was eluted in binding buffer with 2.5 mM D-desthiobiotin, and dialyzed overnight into CSF-XB buffer (10 mM HEPES, 100 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10% sucrose w/v, pH 7.7). 200 μM GTP was included in lysis and elution buffers.
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