The ectodomain of the HLA-A*2402 heavy chain and human β2 microglobulin (β2m) were expressed in Escherichia coli as inclusion bodies and subsequently refolded in vitro in the presence of different peptides. Briefly, the dissolved HLA heavy chain, β2m inclusion body, and peptides were diluted at a molar ratio of 1:1:3, respectively, into a refolding buffer (100 mM Tris-HCl, 400 mM l -arginine, 2 mM EDTA-Na, 5 mM glutathione [GSH], 0.5 mM glutathione disulfide [GSSG]) and slowly stirred for 12 h at 4°C. The refolded complexes were then concentrated and purified by Superdex 200 10/300 GL (GE Healthcare) chromatography, further purified on an ion-exchange Resource Q column (GE Healthcare), and manipulated for crystal screening.
Resource q ion exchange column
Manufactured by GE Healthcare
The Resource Q ion-exchange column is a laboratory equipment used for the purification and separation of biomolecules, such as proteins, peptides, and nucleic acids. It utilizes anion-exchange chromatography principles to isolate and concentrate target analytes from complex mixtures. The column is designed to provide efficient and reproducible separation performance for a wide range of biomolecular applications.
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Lab products found in correlation
Superdex 200 column, by GE Healthcare (3 mentions)
Peristaltic rapid pump, by Gilson (2 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
Smart chromatography system, by GE Healthcare (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Phusion polymerase, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Expicho expression system, by Thermo Fisher Scientific (1 mentions)
Hitrap heparin hp, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Kta purifier hplc system, by GE Healthcare (1 mentions)
Ni nta agarose, by Macherey-Nagel (1 mentions)
Emulsiflex c5 high pressure homogenizer, by Avestin (1 mentions)
8 protocols using resource q ion exchange column
1
In Vitro Refolding of HLA-A*2402 Complex
2
DNA Amplification and Purification Protocol
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The 279 bp linear dsDNA molecules were obtained by PCR amplification of a region of the B. subtilis genome. The 1001 bp and 1876 bp dsDNA molecules were amplified from the pZE14 plasmid (44 (link)). PCRs were done using 5′-phosphorylated primers and the Phusion polymerase (Thermofisher) in order to generate blunt-ended products. For the DNA bridging assay, the one-end biotinylated 1001 bp dsDNA molecule was prepared using one 5′-biotinylated primer and one 5′-phosphorylated primer. Amplified DNA molecules were purified on agarose gel using the QIAquick PCR purification kit (QIAGEN).
The pUC19 plasmid (2686 bp) was isolated from E. coli DH5α cells using Isolating Recombinant Bacmid DNA protocol (Invitrogen) and purified on an ion exchange Resource Q column (GE Healthcare) with a chromatography SMART system (GE Healthcare). For electron microscopy experiments, all DNA molecules were purified on a MiniQ anion exchange column (GE Healthcare) with a chromatography SMART system. The purified DNA was precipitated and resuspended in a 10 mM Tris-HCl, pH 7.5, 1 mM EDTA buffer.
The pUC19 plasmid (2686 bp) was isolated from E. coli DH5α cells using Isolating Recombinant Bacmid DNA protocol (Invitrogen) and purified on an ion exchange Resource Q column (GE Healthcare) with a chromatography SMART system (GE Healthcare). For electron microscopy experiments, all DNA molecules were purified on a MiniQ anion exchange column (GE Healthcare) with a chromatography SMART system. The purified DNA was precipitated and resuspended in a 10 mM Tris-HCl, pH 7.5, 1 mM EDTA buffer.
3
Reconstitution of Histone Octamers and Nucleosomes
4
Recombinant FIBCD1-FReD Protein Expression and Purification
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The cloning, expression, and purification of FIBCD1-FReD have been described in detail (1 (link)). Briefly, for the protein used for the ligand-bound crystal structures, the initial enzyme-linked immunosorbent inhibition assay (ELISA) and the MST affinity assay, FIBCD1-FReD (residues 236–461) was expressed in Sf9 insect cells using the pNT-Bac vector expression system. The recombinant FIBCD1-FReD variants used for the native crystal structure and for the repeated measurements of (GlcNAc)n n = 1, 2, 3 inhibition by ELISA, the same fragments of FIBCD1-FReD (wildtype, H396A, and K381L) were expressed in Chinese hamster ovary (CHO) cells using the ExpiCHO expression system (ThermoFisher Scientific) with FIBCD1-FReD (including the IgG K signal peptide for secretion) cloned into the pcDNA3.1+ plasmid (Genscript). Purification of the FIBCD1-FReD variants, regardless of the expression system, was achieved through affinity chromatography using acetylated Toyopearl AF-Amino-650M resin (Tosoh) followed by ion-exchange chromatography using a Resource Q ion-exchange column (GE Healthcare) as outlined in Schlosser et al. (Ref (1 (link))).
5
Nucleosome Assembly and Purification
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Nucleosome substrates were assembled as described(26 (link), 31 (link)). Equimolar amounts of each lyophilized 4 histones were dissolved in unfolding buffer (6 M guanidinium hydrochloride, 20 mM Tris, pH 7.5, 5 mM DTT), mixed and dialyzed into refolding buffer. Octamers were purified through size exclusion chromatography Superdex 200 column (GE healthcare) in refolding buffer (10 mM Tris, pH 7.5, 2 M NaCl, 1 mM EDTA, 5 mM BME). Nucleosomes were assembled by combining equimolar ratios of purified Widom 601 DNA and histone octamers, and dialyzing the mix overnight with gradient salt dialysis using a peristaltic Rapid Pump (Gilson). Assembled nucleosomes were purified through a Resource Q ion-exchange column (GE Healthcare). Finally, purified nucleosomes were dialyzed into TCS buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT), concentrated, and stored at 4°C until use.
6
Purification of RNA Polymerase Core
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Cell lysate was diluted with TGED (10 mM Tris-HCl pH 7.9, 5% v/v glycerol, 0.1 mM EDTA, and 0.2 mM DTT) with 0.2 M NaCl to about 10 ml of buffer per 1 g of cells. Polyethylenimine was added to 0.6% v/v and centrifuged at 8000 rpm for 10 min. The pellet was then washed with TGED with 0.45 M NaCl. To elute RNAP, TGED with 1 M NaCl was added to the pellet before centrifugation at 8000 rpm for 10 min and the supernatant collected. Ammonium sulphate was then added at 0.35 g/ml of supernatant. This was then centrifuged for 30 min at 15,000 rpm. The pellet was dissolved in TGED with 0.1 M NaCl and loaded on to a HiTrap Heparin HP (GE Healthcare Life Sciences) equilibrated with TGED with no NaCl. A step elution was carried out using TGED with increasing concentrations of NaCl. Fractions containing RNAP were loaded on to a RESOURCE Q ion exchange column (GE Healthcare Life Sciences) pre-equilibrated with TGED with no NaCl. RNAP core was eluted in NaCl gradient (0.15–1 M NaCl) in TGED. Fractions were pooled, concentrated, and supplied with 50% v/v glycerol for storage. σ subunits and GreA factor were cloned from SH1000, expressed with 6xHis tags, and purified by metal-affinity chromatography using standard methods.
7
Purification and Assembly of Nucleosome Substrates
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Nucleosome substrates were assembled as described (27 (link), 32 (link)). Equimolar amounts of each of the four lyophilized histones were dissolved in unfolding buffer [6 M guanidinium hydrochloride, 20 mM tris (pH 7.5), and 5 mM dithiothreitol (DTT)], mixed, and dialyzed into refolding buffer. Octamers were purified through size exclusion chromatography Superdex 200 column (GE Healthcare) in refolding buffer [10 mM tris (pH 7.5), 2 M NaCl, 1 mM EDTA, and 5 mM BME]. Nucleosomes were assembled by combining equimolar ratios of purified Widom 601 DNA and histone octamers and dialyzing the mix overnight with gradient salt dialysis using a peristaltic Rapid Pump (Gilson). Assembled nucleosomes were purified through a Resource Q ion-exchange column (GE Healthcare). Last, purified nucleosomes were dialyzed into dialysis buffer [20 mM tris-HCl (pH 7.5), 1 mM EDTA, and 1 mM DTT], concentrated, and stored at 4°C until use.
8
Recombinant Expression and Purification of PEX5 Variants
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E. coli expression plasmids encoding His6-tagged human PEX5L and PEX5L(1-335) were described previously (Schliebs et al. 1999) . Expression and purification of the His6-tagged PEX5 variants was performed using Ni-NTA-agarose (Co) prepared in empty Chromabond columns for solid-phase extraction (Macherey-Nagel) followed by ResourceQ ion exchange column (GE Healthcare) chromatography using an Äkta purifier HPLC system according to the manufacturer's instructions (GE Healthcare). E. coli lysates were prepared by EmulsiFlex-C5 High Pressure homogenizer (Avestin) in presence of protease inhibitor mix containing 8 mM Antipain, 0.3 mM Aprotinin, 1 mM Benzamidin hydrochloride, 1 mM Bestatin, 10 mM Chymostatin, 5 mM Leupeptin, 5 mM sodium fluoride, 15 mM Pepstatin A and 1 mM phenymethylsulfonylfluoride. The lysate and all washing and elution buffers at pH 8.0 were supplemented with 1 mM dithiothreitol.
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