The Champion™ pET SUMO expression vector (K30001) and PureLink™ Quick Gel Extraction Kit (K210012) were purchased from Invitrogen. DreamTaq Green PCR Master Mix (K1082), T4 DNA ligase (5U/µl), Rapid DNA Ligation Kit, GeneJET Plasmid Miniprep Kit, isopropyl β-D-1-thiogalactopyranoside (IPTG), and PageRuler™ Unstained Protein ladder were obtained from Thermo Scientific™. Rosetta 2(DE3), Rosetta 2(DE3) pLysS, and Rosetta-gami™ 2(DE3) cells were purchased from Novagen, and HepG2, HCT-116, and MCF-7 cell lines for the biological assay were purchased from ATCC, USA. Terrific broth and tetracycline hydrochloride were purchased from Fisher bioreagents™, USA. Streptomycin sulfate, chloramphenicol, and a purification column (Ni-Sepharose column) were purchased from Sigma. The Ni-Sepharose™ 6 Fast Flow resin was purchased from GE Healthcare Life Sciences. Kanamycin monosulfate was obtained from ICN Biomedicals, Inc. IL28/29 (H-1) sc-365834 mouse monoclonal IgG2a antibody and goat anti-mouse IgG-AP-conjugated antibody were purchased from Santa Cruz Biotechnology. The Rainbow™ Full-range Molecular Marker (Mr 12,000–225,000) was purchased from GE Healthcare Life Sciences. DMEM medium, fetal bovine serum, penicillin–streptomycin and trypsin were purchased from Gibco.
Ni sepharose column
Manufactured by Merck Group
Ni-Sepharose column is a chromatography resin used for the purification and isolation of histidine-tagged recombinant proteins. The resin is composed of nickel-charged agarose beads that selectively bind to the histidine tag on the target protein, allowing it to be separated from other components in the sample.
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Lab products found in correlation
Ni sepharose 6 fast flow resin, by GE Healthcare (2 mentions)
Tetracycline hydrochloride, by Thermo Fisher Scientific (1 mentions)
Streptomycin sulfate, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Purelink quick gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Terrific broth, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Pageruler unstained protein ladder, by Thermo Fisher Scientific (1 mentions)
Rapid dna ligation kit, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Kta system, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 column, by Cytiva (1 mentions)
3 protocols using ni sepharose column
1
Recombinant Protein Expression and Purification
2
Purification of Native Interleukin-29
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Single step purification of native IL-29 was done by affinity chromatography on ÄKTA system (GE Healthcare, USA). SUMO digested sample was loaded on Ni-Sepharose column (Sigma) packed with Ni-Sepharose™ 6 Fast Flow resin (GE Healthcare Life Sciences™) on ÄKTA system. Native IL-29 protein was obtained through flow-through. Furthermore, the uncleaved fusion protein and SUMO tag were eluted using 800 mM imidazole. The protein fraction was quantified using the Bradford assay and analyzed using 12% SDS-PAGE. Bradford assay was performed by taking the absorbance of purified IL-29 and the known standards of 1–5 mg/ml BSA solution and comparing the concentration of IL-29 with the known concentration of BSA. The fraction containing native IL-29 protein was buffer-exchanged using PD-10 desalting column (GE Healthcare) pre-packed with Sephadex G-25 and subjected to further protein characterization.
3
Cloning and Purification of GbGtfC Protein
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The cloning and expression
of the GbGtfC-ΔC construct, containing residues 33–738
of Geobacillus 12AMOR1 GtfC and a 20-residue N-terminal
His-tag, have been described before.22 (link) Briefly,
the pET15b vector carrying the gtfC construct was
overexpressed in E. coli BL21 (DE3)
cultures grown at 37 °C; harvested cells were resuspended and
broken by sonication; cell-free extract (CFE) was stored at 4 °C.
The GbGtfC-ΔC protein in the CFE was captured by immobilized
metal affinity chromatography (IMAC) on a Ni-Sepharose column (Sigma-Aldrich,
St. Louis, MO) using an elution buffer containing 20 mM Tris-HCl,
pH 8.0, 100 mM NaCl, and 350 mM imidazole. Fractions with the highest
absorbance at 280 nm were pooled and concentrated using a VivaSpin
4 (molecular weight cutoff 10 kDa) at 4000g. The
final purification step was done via size exclusion
chromatography on an Äkta Micro system equipped with a Superdex
200 Increase 10/300 column (Cytiva, Marlborough, MA) at 12 °C.
The elution buffer contained 20 mM MES-NaOH, pH 6.1, 100 mM NaCl,
and 1 mM CaCl2. The center fractions of the peak eluting
at 13.3–14.8 mL were pooled (Figure S1 ) and concentrated as described above to obtain the final GbGtfC-ΔC
protein sample suitable for crystallization. Protein concentrations
were determined by measuring the absorbance at 280 nm using a NanoDrop
One spectrophotometer (Isogen Life Science, De Meern, The Netherlands).
of the GbGtfC-ΔC construct, containing residues 33–738
of Geobacillus 12AMOR1 GtfC and a 20-residue N-terminal
His-tag, have been described before.22 (link) Briefly,
the pET15b vector carrying the gtfC construct was
overexpressed in E. coli BL21 (DE3)
cultures grown at 37 °C; harvested cells were resuspended and
broken by sonication; cell-free extract (CFE) was stored at 4 °C.
The GbGtfC-ΔC protein in the CFE was captured by immobilized
metal affinity chromatography (IMAC) on a Ni-Sepharose column (Sigma-Aldrich,
St. Louis, MO) using an elution buffer containing 20 mM Tris-HCl,
pH 8.0, 100 mM NaCl, and 350 mM imidazole. Fractions with the highest
absorbance at 280 nm were pooled and concentrated using a VivaSpin
4 (molecular weight cutoff 10 kDa) at 4000g. The
final purification step was done via size exclusion
chromatography on an Äkta Micro system equipped with a Superdex
200 Increase 10/300 column (Cytiva, Marlborough, MA) at 12 °C.
The elution buffer contained 20 mM MES-NaOH, pH 6.1, 100 mM NaCl,
and 1 mM CaCl2. The center fractions of the peak eluting
at 13.3–14.8 mL were pooled (
protein sample suitable for crystallization. Protein concentrations
were determined by measuring the absorbance at 280 nm using a NanoDrop
One spectrophotometer (Isogen Life Science, De Meern, The Netherlands).
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