For the expression of Arabidopsis GPX1 and GPX7, the corresponding cDNAs, excluding the predicted transit peptides and the stop codons, were amplified with iProof™ High-Fidelity DNA Polymerase (Bio-Rad) using oligonucleotides listed in Table S1 , which added NcoI and XhoI sites at the 5’ and 3’ ends, respectively. For the expression of Arabidopsis TRX m4, the corresponding cDNA, excluding the predicted transit peptides and including the stop codon, was amplified as described above using oligonucleotides (Table S1 ) adding BamHI and HindIII sites at the 5’ and 3’ ends, respectively. PCR products were gel-purified, cloned in pGEMt vector (Promega), and sequenced. For GPXs, pGEMt-derived plasmids were digested with NcoI and XhoI, subcloned in the pET28 (Qiagen) expression vector and introduced into E. coli BL21 (DE3) cells. The pGEMt-TRX m4 plasmid was digested with BamHI and HindIII, subcloned in the pQE30 (Qiagen) expression vector and introduced into E. coli XL1Blue. Over-expressed recombinant proteins, containing a His-tag at the C-terminus (GPX1 and GPX7) or the N-terminus (TRX m4), were purified by nitrile triacetic acid (NTA) affinity chromatography in Hi-Trap affinity columns (GE Healthcare). Recombinant His-tagged NTRC and 2-Cys PRX from rice [38 (link)] and TRXs of the types y2, x and f2 from Arabidopsis were obtained as previously described [31 (link),41 (link)].
Hi trap affinity columns
Manufactured by GE Healthcare
Sourced in France, United States
Hi-Trap affinity columns are a type of lab equipment used for protein purification. They are designed to capture and isolate specific target proteins from complex samples through the process of affinity chromatography. The columns contain a matrix with immobilized ligands that selectively bind to the target proteins, allowing them to be separated from other components in the sample.
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Lab products found in correlation
Hitrap, by Cytiva (3 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
Pgem t vector, by Promega (1 mentions)
Pqe30, by Qiagen (1 mentions)
Pet28, by Qiagen (1 mentions)
Quickchange 2 xl kit, by Agilent Technologies (1 mentions)
Oligonucleotides, by Eurogentec (1 mentions)
Anti flag m1 agarose affinity gel, by Merck Group (1 mentions)
Bl21 codonplus de3 cells, by Thermo Fisher Scientific (1 mentions)
Akta 900 fast protein liquid chromatography, by GE Healthcare (1 mentions)
Gst trap hp columns, by GE Healthcare (1 mentions)
5 protocols using hi trap affinity columns
1
Recombinant Expression and Purification of Arabidopsis Thioredoxins
2
Recombinant Arabidopsis 2-Cys Prx Purification
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Recombinant 2-Cys Prxs A and B from Arabidopsis were produced in Escherichia. coli XL1-Blue with a His-tag at the N-terminus, as previously described (Kirchsteiger et al., 2009 (link)). Recombinant proteins were purified from crude extracts of E. coli cultures by chromatography in pre-packed Hi-Trap affinity columns (GE Healthcare). Recombinant proteins, 2-Cys Prxs A and B, at a concentration of 0.1 µg µl–1 in 100mM phosphate buffer (pH 7.4), 0.5M NaCl, and 10% (v/v) glycerol, were incubated for 30min with or without 20mM DTT and then for another period of 30min with increasing concentrations of H2O2 (0.1–10mM). Protein samples (0.125 µg of protein) were subjected to SDS-PAGE, under reducing or non-reducing conditions, blotted on nitrocellulose membranes and probed with anti-SO2/3 antibody.
3
Purification and Characterization of Complement Proteins
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Oligonucleotides were purchased from Eurogentec, restriction and modification enzymes from New England Biolabs. C1s Fg40 and pEDS variants were engineered using site-directed mutagenesis with the QuickChange II XL kit (Agilent Technologies). Nα-Benzoyl-L-arginine ethyl ester hydrochloride (Bz-Arg-OEt, BAEe) and ANTI-FLAG® M1 Agarose Affinity Gel were purchased from Sigma-Aldrich. HiTrap affinity columns were from GE Healthcare Life sciences, France. The following complement proteins were purified from human plasma according to published procedures (8 (link), 9 (link)): C4, C1 inhibitor and activated C1r. For protein quantification, the following absorbance coefficient A1%, 1cm at 280 nm and molecular weight (Mw) were used: C1r (12.4 and 86,300), C4 (8.3 and 205,000), C1inh (4.5 and 104,000) (8 (link), 10 (link)), respectively.
4
Purification and Antibody Production of Drosophila GOLPH3
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GST-full-length Drosophila GOLPH3 (dGOLPH3) proteins and GST-full-length Drosophila Rab11 were expressed in BL21-CodonPlus [DE3] cells (Invitrogen) and purified using HiTrap affinity columns (GTtrap FF, and GSTtrap HP columns, GE Healthcare) operated with AKTA 900 Fast Protein Liquid Chromatography. Polyclonal antisera were raised against the purified GST-dGOLPH3 protein. Immunization was carried out at Agro-Bio Services (www.agro-bio.com ); two rabbits and two mice were injected using standard procedures. The anti-GST-GOLPH3 antisera were first depleted of anti-GST antibodies and then affinity-purified against GST-GOLPH3.
5
Recombinant Ts-CLP Protein Purification
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The sequence of Ts-clp (GenBank: EU263325.1) without the N-terminal signal peptide was amplified from the cDNA of L6h by PCR with primers (forward, 5′-CCG AAT TCC AGA TAC TTG GTG A-3′, containing the EcoRI restriction site; reverse, 5′-TAG CTC GAG TTA ACG GAA AAA AGT GA-3′, containing the XhoI restriction site). The PCR products were subcloned into the expression vector pET-28a with EcoRI and XhoI sites. After transformation into Escherichia coli BL21 (DE3) cells (Novagen, Germany), the expression of rTs-CLP was induced with 1 mM IPTG for 6 h at 37°C. The bacterial culture pellet was resuspended in solution (20 mM Tris–HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100). Lysozyme was added to the solution and the mixture was incubated at 37°C for 15 min and then sonicated on ice. Following centrifugation at 3 000 rpm, the soluble and insoluble fractions were obtained and analysed by 12% SDS-PAGE. The proteins were purified using HiTrap™ affinity columns (GE healthcare, USA) according to the manufacturer’s instructions.
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