For the protein purification of the CLC-7/Ostm1 complex, 2 liters of transfected HEK293F cells was harvested by centrifugation at 3000g. Cell pellets were resuspended in lysis buffer containing 20 mM Hepes (pH 7.4) and 150 mM NaCl, leupeptin (1 μg/ml), pepstatin (1.5 μg/ml), aprotinin (0.84 μg/ml), 0.3 mM phenylmethylsulfonyl fluoride and lysed by sonication for 5 min. The cell membrane was pelleted after a 100,000g ultracentrifugation for 1 hour. The membrane was resuspended in buffer containing 20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM dithiothreitol (DTT), and 1% (w/v) digitonin for 2 hours with gentle rotation at 4°C. After ultracentrifugation at 100,000g for 20 min, the supernatant was incubated with Strep-Tactin Sepharose (IBA) for 1 hour with gentle rotation at 4°C. The resin was washed extensively with wash buffer containing 20 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM DTT, and 0.1% (w/v) digitonin. The target CLC-7/Ostm1 complex was eluted with wash buffer plus 5 mM d -desthiobiotin (IBA) and concentrated to a final volume of approximately 100 μl. The final protein was applied to size-exclusion chromatography (Superpose-6 10/300 GL, GE Healthcare) in buffer containing 20 mM Hepes (pH 7.4), 150 mM NaCl, and 0.1% digitonin. The peak corresponding to the CLC-7/Ostm1 complex was collected for further cryo-microscopy analysis.
Superpose 6 10 300 gl
Manufactured by GE Healthcare
The Superpose-6 10/300 GL is a size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. It features a bed volume of 24 mL and a fractionation range suitable for separating molecules with molecular weights between 5,000 and 500,000 Daltons.
Automatically generated - may contain errors
Lab products found in correlation
Ecori, by GenScript (1 mentions)
Hindiii, by GenScript (1 mentions)
Complete his tag purification resin, by Roche (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
D desthiobiotin, by GE Healthcare (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Nanodrop, by Implen (1 mentions)
4 protocols using superpose 6 10 300 gl
1
Purification of CLC-7/Ostm1 Protein Complex
2
Purification and Labeling of Pentameric GLIC Receptor
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MBP-GLIC was produced as previously described (Bocquet et al., 2009 (link)), in BL21 E.Coli strains. Briefly, MBP-GLIC was produced at 20° C in BL21 bacteria after IPTG (20 mM) induction. After cell disruption by sonication, membranes were separated through ultracentrifugation (40,000 rpm) and MBP-GLIC extracted in buffer A 2% DDM overnight. MBP-GLIC was purified on an amylose resin and eluted by maltose addition in buffer A 0.02% DDM. Proteins were run on a gel filtration (superpose 6 10/300 GL (GE Healthcare, Chicago, IL)) column for removal of remaining maltoporin contaminants. The MBP was subsequently cleaved from GLIC by addition of thrombine (Merck Millipore, Billerica, MA) overnight, and GLIC purified again through gel filtration in buffer A 0.02% DDM. For receptors with the K33C and E243C mutations, the protein was incubated for 1 hr with 10 mM DTT prior to the gel filtration to reduce potential disulfide bridges. Pentameric GLIC was then incubated with mBBr at a five molar excess ratio (monomer 1:5 mBBr) while ensuring the final DMSO concentration did not exceed 1%, overnight, under agitation, at 4°C. Excess fluorophore was removed by gel filtration and GLIC-labeled samples flash frozen for storage at −80°C.
3
Optimized CD4i Fusion Protein Production
4
Purification of ABCB6 Membrane Protein
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For the protein purification of the ABCB6, two liters of transfected HEK293F cells were harvested by centrifugation at 3000× g. Cell pellets were resuspended in lysis buffer containing 20 mM Hepes, pH 7.4, and 150 mM NaCl, a protease inhibitor cocktail tablet (Roche, 1:1000), and lysed by sonication for 5 min. The cell membrane was pelleted after a 100,000× g ultracentrifugation for 1 h. The membrane was resuspended in a buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM DTT, and 1% (w/v) C12E9 for 1.5 h with gentle rotation at 4 °C. After ultracentrifugation at 100,000× g for 15 min, the supernatant was incubated with Strep-Tactin Sepharose (IBA) for 0.5 h with gentle rotation at 4 °C. The resin was washed extensively with wash buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, and 0.01% (w/v) C12E9. The target protein was eluted with wash buffer plus 5 mM d -desthiobiotin (IBA) and concentrated to a final volume of approximately 100 μL. The final protein was applied to size-exclusion chromatography (Superpose-6 10/300 GL, GE Healthcare) in a buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, and 0.01% C12E9. The peak corresponding to the ABCB6 or ABCB6 mutant was collected and protein concentrations in detergent micelles were measured by absorbance at 280 nm using a NanoDrop (Implen).
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