The chimeric protein-codifying gene sequence was commercially synthetized as having 1107 bp and it was cloned into prokaryotic expression vector pET28a-TEV (Genscript®, Piscataway, NJ, USA). The construct was transformed into Escherichia coli BL-21 strain cells, and the protein was expressed by addition of 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Promega, Madison, WI, USA) for 2 h at 37 °C in 1 L of LB medium (with yield of 36 mg/L of culture). Next, bacteria were centrifuged at 3000× g for 10 min at 4 °C and suspended in lysis buffer (20 mM Tris-HCL, pH 8.0; 0.5 M NaCl; 5 mM imidazole; 8 M urea; 1 mM β-mercaptoetanol), followed by six cycles of ultrasonication for 30 s each (at 90 Hz). Cellular debris was removed after 5000× g centrifugation for 15 min at 4 °C and the supernatant was collected. The N4S11-SC2 protein was purified on a HisTrap HP affinity column (GE Healthcare Life Sciences, Chicago, IL, USA) connected to an AKTA system. Inclusion bodies were solubilized in 2 M urea buffer. A 12.5% (w/v) SDS-PAGE was done to evaluate the purity of the recombinant protein. A Page Ruler broad range unstained protein ladder (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) was used.
Histrap hp affinity column
Manufactured by GE Healthcare
Sourced in United States, Sweden, China
The HisTrap HP affinity column is a chromatography column designed for the purification of histidine-tagged recombinant proteins. It contains a resin with immobilized nickel ions that bind to the histidine tag, allowing the target protein to be separated from other components in the sample.
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Lab products found in correlation
Hitrap, by Cytiva (5 mentions)
Hitrap q hp column, by GE Healthcare (4 mentions)
Kta pure protein purification system, by GE Healthcare (4 mentions)
Pd 10 desalting column, by GE Healthcare (2 mentions)
Pet28a tev, by GenScript (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Promega (1 mentions)
Superdex 200 gel filtration column, by GE Healthcare (1 mentions)
Akta system, by GE Healthcare (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Polymyxin agarose column, by Merck Group (1 mentions)
Pet28a tev vector, by GenScript (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Pevol pbpf, by Addgene (1 mentions)
Protein sds page loading buffer, by Takara Bio (1 mentions)
Bicinchoninic acid assay, by Sangon (1 mentions)
Hiload 16 60 superdex 200 prep grade column, by GE Healthcare (1 mentions)
Glucose assay kit, by Merck Group (1 mentions)
Akta purifier system, by GE Healthcare (1 mentions)
Bl21codon plus rp, by Agilent Technologies (1 mentions)
27 protocols using histrap hp affinity column
1
Recombinant N4S11-SC2 Protein Production
2
Purification of Recombinant Protein EC12
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Soluble proteins were collected after centrifugation at 18,000 rpm for 30 min at 4°C and filtered. Subsequently, protein purification was carried out with a HisTrap HP affinity column (GE Healthcare Life Sciences) equilibrated with cell re-suspension buffer on an ÄKTA pure protein purification system (GE Healthcare Life Sciences). Elution of proteins was carried out in a gradient of equilibration buffer containing 20 mM Tris buffer (pH 8.0), 300 mM NaCl, 2 mM DTT and 1 M imidazole. Fractions containing EC12 were subjected to western blot analysis using anti-6xHis antibody. The fractions were pooled and separated on a HiTrap Q HP column (GE Healthcare Life Sciences) equilibrated with buffer containing 20 mM Tris (pH 8.0), 2 mM DTT and eluted in a gradient from 0 to 1 M NaCl. At the last step, the 6xHis tag on the recombinant protein was removed by a protease encoded by the TEV. HisTrap chromatography was then used to separate untagged recombinant proteins. The purified protein was concentrated and frozen for future use. EC12 mutants used in the competitive binding assay were obtained through the first HisTrap affinity purification without TEV cleavage.
3
Recombinant ChimT Protein Expression
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The gene encoding ChimT was commercially synthesized in the pET28a-TEV vector by GenScript® (Piscataway, NJ, USA). The recombinant protein was expressed in E. coli Artic Express cells (DE3, Agilent Technologies, Santa Clara, CA, USA) adding 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich, St. Louis, MO, USA), with shaking at 100 rpm for 24 h at 12 °C. The bacterial cells were ruptured by five cycles of ultrasonication of 30 s. each (38 MHz) followed by six cycles of freezing and thawing. Debris was removed by centrifugation and ChimT was passed over a HisTrap HP affinity column connected to an AKTA system (GE Healthcare, Boston, MA, USA) and further purified on a Superdex™ 200 gel-filtration column (GE Healthcare Life Sciences, Boston, MA, USA). The purified protein was then passed over a polymyxin-agarose column (Sigma-Aldrich, St. Louis, MO, USA) to remove any residual endotoxin content: less than 10 ng of lipopolysaccharide per 1 mg of protein was detected with the Quantitative Chromogenic Limulus Amebocyte Kit (QCL-1000 model, BioWhittaker, Walkersville, MD, USA) following the manufacturer’s instructions.
4
Purification of Recombinant Proteins with Affinity and Ion Exchange Chromatography
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Soluble proteins were collected after centrifugation at 18,000 rpm for 30 min at 4°C and filtered. Protein purification was carried out with a HisTrap HP affinity column (GE Healthcare Life Sciences) equilibrated with cell re-suspension buffer on an ÄKTA pure protein purification system (GE Healthcare Life Sciences). Elution of proteins was carried out in a gradient of cell re-suspension buffer and 20 mM Tris buffer pH 8.0 supplemented with 300 mM NaCl, 2 mM DTT and 1 M imidazole. Fractions containing EC12 were pooled and separated on a HiTrap Q HP column (GE Healthcare Life Sciences) equilibrated with 20 mM Tris pH 8.0 plus 2 mM DTT and eluted in a gradient of 20 mM Tris pH 8.0 supplemented with 1 M NaCl and 2 mM DTT. At the last step, the 6xHis tag on the recombinant protein was removed by a protease encoded by the TEV. HisTrap chromatography was used to separate untagged recombinant proteins. The purified protein was concentrated and frozen for future use.
5
Recombinant Pullulanase Production in E. coli
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E. coli JM109 and E. coli BL21 (DE3) were used as the hosts for recombinant gene cloning and expression, respectively. The recombinant plasmid pET28a-Btpul (GenBank Accession No. EMBL AJ315595) was constructed by Genewiz (Suzhou, China). The plasmid pEVOL-pBpF encoding AARS/tRNA was purchased from Addgene (Beijing, China). PrimeSTAR HS DNA polymerase, which was used for polymerase chain reaction (PCR), was purchased from TaKaRa Biotechnology Co., Ltd. (Beijing, China). Primers were all synthesized by Genewiz (Suzhou, China). The ClonExpress II One Step Cloning Kit for homologous recombination was purchased from the Vazyme Biotech Company, Ltd. (Nanjing, China). DNA sequencing was completed by Genewiz (Suzhou, China). The HisTrap HP affinity column and disposable PD-10 desalting column that were respectively used for protein purification and desalination were purchased from GE Healthcare Life Sciences (Beijing, China). O2beY, pullulan, and l -arabinose were purchased from Tokyo AMATEK (Suzhou, China), Chemical Industry (Tokyo, Japan), and Ourchem (Shanghai, China), respectively. Premixed Protein Marker (High) and 4× Protein SDS-PAGE Loading Buffer were purchased from TaKaRa Biotechnology Co., Ltd. (Beijing, China). All other materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Sinopharm Chemical Reagent (Shanghai, China).
6
Purification of SARS-CoV-2 nsp14-nsp10 Complex
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The protein was purified as previously [10 (link)]. Briefly, transformed Escherichia coli BL21Gold (DE3) cells with single pRSF-duet-1 plasmid bearing C-terminal 6xHis-tagged full-length nsp14 and nsp10 were grown in LB media. The protein expression was induced by adding 0.5mM IPTG and incubated at 15°C for 18hours. The cells were harvested and resuspended in binding buffer (25mM Tris pH 7.5, 250mM NaCl, 10% glycerol, 0.01% IGEPAL, 25mM imidazole, 10μM ZnCl2 and 10mM 2-mercaptoethanol). The cells were lysed by sonication and the supernatant was loaded onto a HisTrap HP affinity column (GE Healthcare). The column was washed with binding buffer and then the complex was eluted using buffer with 500mM imidazole. The eluted protein was further subjected to size exclusion chromatography with a buffer containing 100mM KH2PO4/K2HPO4 buffer pH 8.0, 100mM KCl, 0.01% IGEPAL, 5mM 2-mercaptoethanol and 10% Glycerol.
7
Affinity Purification of Protein Complexes
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Before starting these assays, the 6His‐RedR1, 6His‐RedR2, or MBP‐6His‐BtdS proteins were purified as described in the previous section. After dialysis against buffer B without imidazol, the assay started by binding the purified protein to a HisTrap HP affinity column (GE Healthcare). Then a crude extract containing the overproduced protein of interest (RedR1‐HA or RedR2‐HA) was obtained by 3 passages of 1 L culture of either BL21 (pET24bRedR1HA) or BL21 (pET24bRedR2HA) concentrated in 30 mL of buffer A through a French pressure cell. The crude extract was centrifuged at 13 000 g for 1 h at 4 °C. The resulting supernatant containing the HA‐tagged protein was loaded onto the column where the 6His‐RedR1, 6His‐RedR2, or MBP‐6His‐BtdS protein had been previously loaded, then the column was washed with up to 10 column volumes of buffer A. The bound proteins were then eluted in buffer B supplemented with 250 mm imidazole. Fractions (2 mL) were collected throughout the process and analysed by western blot with either anti‐His or anti‐HA antibodies to identify each protein.
8
Recombinant Alg2 Protein Expression
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All Alg2 proteins used in this study expressed in E. coli contain an N-terminal His6-Trx tag. A series of E. coli pET32 expression plasmids, containing ScAlg2, ScAlg2G257P, hAlg2, ScAlg1ΔTM, and hMGAT1ΔTM genes were transformed into E. coli Rosetta (DE3) cells (Merck) and cultured for expression of recombinant proteins. Recombinant proteins were extracted and purified with a HisTrap HP affinity column (GE Healthcare Life Sciences), as described previously19 (link). Purified proteins were analyzed by SDS-PAGE, followed by staining with Coomassie Brilliant Blue R-250. Protein concentration was determined by the bicinchoninic acid assay (Sangon Biotech).
9
Purification and Characterization of Recombinant Receptors
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Full length of MgR, McR, MhR, EcR and RNase II were cloned into the expression vector pET19b containing an N-terminal 10× His-tag, respectively. MgR and EcR mutants were generated by quick-change mutagenesis kits (Agilent). Constructs were verified by DNA sequencing (Tsing Ke Biotech). The recombinant plasmids were transformed into BL21 (DE3) E. coli cells and induced with 0.2 mM IPTG in Terrific Broth media at 16°C overnight. The resulting His-tagged protein was purified by a HisTrap HP affinity column (GE Healthcare), eluted in 50 mM Tris (pH 7.5), 500 mM NaCl, 5 mM MgCl2, 5% (vol/vol) glycerol and 300 mM imidazole. The protein was further purified using a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with 50 mM Tris (pH 7.5), 300 mM NaCl, 5 mM MgCl2 and 10% (vol/vol) glycerol. The protein purify was examined by SDS-PAGE. Proteins were pooled and concentrated to 15 mg/ml, flash frozen in liquid nitrogen and stored at −80°C.
10
Large-scale EGFP and PΙNP Production
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To validate large-scale expression of EGFP of the top three strongest vectors and achieve large-scale production of PΙNP. After overnight activation, 200 mL of C. glutamicum seed solutions were all transferred to 1.8 L of LBB medium (30 g/L glucose) in a 5 L fermenter (Applikon EZ-control). Throughout the total 48 h of cultivation, the temperature was maintained at 30 °C. The dissolved oxygen was maintained at 30% (v/v). The speed was set to 400–1000 r/min and the pH of the medium was controlled at 7. To avoid glucose starvation, 50 mL glucose solution (300 g/L) was added every 4 h after 12 h of inoculation. Glucose concentrations in the culture medium were monitored by a glucose assay kit (Sigma, St. Louis, Missouri, USA).
The protein purification steps were presented below: the medium supernatant contained PΙNP were collected first, protein purification used an AKTA purifier system (GE, Sweden) and a HisTrap HP affinity column. Protein quantity and purity was determined by SDS-PAGE analysis.
The protein purification steps were presented below: the medium supernatant contained PΙNP were collected first, protein purification used an AKTA purifier system (GE, Sweden) and a HisTrap HP affinity column. Protein quantity and purity was determined by SDS-PAGE analysis.
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