The CbSITL construct was expressed in the protease‐deficient S. cerevisae strain FGY217 (MATα, ura3‐52, lys2Δ201, pep4Δ) and then purified in a manner described by Knight et al. (2016 ). Briefly, yeast cells were harvested, lysed and cell membranes were sedimented after centrifugation at 180,000g for 1 h. Membranes were resuspended in 50 mM TrisHCl, pH 7.4, 150 mM NaCl, 5% glycerol and homogenized. Proteins were solubilized using 2% FC‐12 and CbSITL was then applied to a 1 ml ‘HisTrap’ Ni2+ column (GE Healthcare, Chalfont St Giles, UK) equilibrated in column buffer (50 mM TrisHCl, pH 7.4, 150 M NaCl, 5% glycerol, + 0.1% FC‐12) plus 20 mM imidazole. The column was washed with 40 ml of Column Buffer plus 60 mM imidazole, and then CbSITL was eluted in Column Buffer with 0.5 M imidazole. Imidazole was immediately removed by gel filtration. Recombinant CbSITL was used to prepare proteoliposomes.
Histrap ni2 column
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The HisTrap' Ni2+ column is a chromatography column used for the purification of histidine-tagged proteins. It contains a Ni-NTA agarose resin that binds to the histidine tag on the target protein, allowing for its separation from other components in the sample.
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Lab products found in correlation
3 protocols using histrap ni2 column
1
Purification of Recombinant CbSITL Protein
2
Purification of Wild-type and Recombinant Transglutaminase
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For the wild-type TGase, the supernatant was collected by centrifugation and concentrated by an ultrafiltration membrane of 10 kDa (molecular weight) four times at 10−15 °C (Lu et al., 2003 (link)). Pre-cooled ethanol was added (final concentration = 60%, v/v) and the precipitate was obtained by centrifugation. The precipitate was dissolved with a 20 mM Tris-HCl buffer (pH 8.0), which was centrifuged at 8000×g for 10 min to remove the insoluble matter, and then filtered using a 0.22-μm membrane. TGase was purified by an SP70 ion exchange column. The eluent buffer was a 20 mM Tris-HCl buffer (pH 8.0) with 1.0 M NaCl, and TGase was purified by desalting with a Superdex 75 column (GE Healthcare, New York, USA).
For recombinant TGase with a His 6 tag, the supernatant was collected by centrifugation. A His-Trap Ni2+ column (GE Healthcare, New York, USA) was used for purification of recombinant TGase, and 20 mM Na2HPO4–NaH2PO4 buffer (pH 7.4) with 50 mM NaCl and 300 mM imidazole was used for the eluent buffer. TGase was purified by desalting with a Superdex 75 column (GE Healthcare, New York, USA).
For recombinant TGase with a His 6 tag, the supernatant was collected by centrifugation. A His-Trap Ni2+ column (GE Healthcare, New York, USA) was used for purification of recombinant TGase, and 20 mM Na2HPO4–NaH2PO4 buffer (pH 7.4) with 50 mM NaCl and 300 mM imidazole was used for the eluent buffer. TGase was purified by desalting with a Superdex 75 column (GE Healthcare, New York, USA).
3
Purification of H-NS C21S Mutant
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H-NS C21S was purified as described for non-tagged H-NS in (Boudreau et al., 2018) (link) with the following modifications. Briefly, BL21 λDE3 cells transformed with pHNSC21S and pHiC were grown in 2 L of LB supplemented with ampicillin (100 µg/mL) + gentamycin (10 µg/mL) to OD600 ~0.6 before inducing expression of H-NS C21S with 0.5 mM isopropyl β-D-1-1/19/2021 thiogalactopyranoside (IPTG) for 2 h at 37 °C. Pelleted cells were lysed by sonication. H-NS C21S was precipitated with addition of 0.6% (w/v) PEI and eluted with PEI elution buffer containing 0.8 M NaCl followed by ammonium sulfate precipitation. Precipitated H-NS was dissolved in nickel binding buffer and applied to a HisTrap Ni 2+ column (GE Healthcare).
Fractions containing H-NS were mixed with TEV protease to remove the N-terminus His6x-tag.
Fractions containing H-NS were mixed with TEV protease to remove the N-terminus His6x-tag.
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