The oligonucleotides used in this study were purchased from Takara (Dalian, China). Escherichia coli strain DH5α was used for gene cloning, and the Rosetta2(DE3)pLysS strain was used for recombinant protein expression. The expression vectors pET28a and pDEST17 were used to express recombinant proteins. The H. volcanii strain and plasmids for gene knockout were provided by Dr Allers (University of Nottingham, UK). Pyrococcus furiosus genomic DNA was purchased from the American Type Culture Collection (ATCC). KOD-plus DNA polymerase was purchased from Toyobo. Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad. The crosslinking reagent BS3 and the DNA damage reagents 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), and methyl methanesulfonate (MMS), methyl methanesulfonate (MMS), N3-methyladenine and adenine were purchased from Sigma–Aldrich (St Louis, MO). All other chemicals and reagents were of analytical grade.
Nickel nitrilotriacetic acid resin
Manufactured by Bio-Rad
Sourced in United States
Nickel–nitrilotriacetic acid resin is an affinity chromatography resin used for the purification of histidine-tagged proteins. It consists of nickel ions chelated to nitrilotriacetic acid, which bind to the histidine tags of recombinant proteins, allowing their separation from other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Kod plus dna polymerase, by Toyobo (4 mentions)
Oligodeoxyribonucleotide, by Thermo Fisher Scientific (2 mentions)
Oligoribonucleotides, by Thermo Fisher Scientific (2 mentions)
Rnase a inhibitor, by Takara Bio (2 mentions)
4 nitroquinoline 1 oxide 4nqo, by Merck Group (1 mentions)
Oligonucleotides, by Takara Bio (1 mentions)
Mitomycin c mmc, by Merck Group (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Kta purifier system, by Cytiva (1 mentions)
Pdest17, by Thermo Fisher Scientific (1 mentions)
Table s1, by Thermo Fisher Scientific (1 mentions)
Primstar max dna polymerase, by Takara Bio (1 mentions)
7 protocols using nickel nitrilotriacetic acid resin
1
Recombinant Protein Expression in E. coli and H. volcanii
2
Recombinant Dbh Expression and Characterization in S. acidocaldarius
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The
S.
acidocaldariusstrain used in this study was provided by Professor Sonja-Verena Albers from the Laboratory of Molecular Biology of Archaea, Institute of Biology, University of Freiburg (Freiburg, Germany). Its genomic DNA was extracted by using phenol-chloroform. The expression vector pDEST17 was used to express recombinant Dbh. The Escherichia coli strains DH5α and Rosetta2(DE3)pLysS were used for gene cloning and recombinant protein expression, respectively. PrimeSTAR Max DNA polymerase was purchased from TaKaRa (Shiga, Japan). Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad (Hercules, USA). The DNA oligonucleotide substrates used for biochemical characterization of Dbh activity are shown in
Supplementary Table S1 and were synthesized by Biosune (Shanghai, China). All other chemicals and reagents were of analytical grade.
S.
acidocaldariusstrain used in this study was provided by Professor Sonja-Verena Albers from the Laboratory of Molecular Biology of Archaea, Institute of Biology, University of Freiburg (Freiburg, Germany). Its genomic DNA was extracted by using phenol-chloroform. The expression vector pDEST17 was used to express recombinant Dbh. The Escherichia coli strains DH5α and Rosetta2(DE3)pLysS were used for gene cloning and recombinant protein expression, respectively. PrimeSTAR Max DNA polymerase was purchased from TaKaRa (Shiga, Japan). Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad (Hercules, USA). The DNA oligonucleotide substrates used for biochemical characterization of Dbh activity are shown in
3
Purification of Hexahistidine-Tagged PaHigA
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Bacterial cells were grown to mid-log phase (OD600 nm = ∼0.8) in LB media at 37°C in the presence of 50 μg/mL Kanamycin and 100 μg/mL chloroamphenicol. Induction of the culture was then carried out with 0.3 mM isopropyl-1-thio-β-D -galactopyranoside (IPTG) at 20°C. Cells were pelleted after 20 h by centrifugation at 8, 000 rpm for 10 min at 4°C. The cell pellet was resuspended in buffer A [20 mM Tris, 300 mM NaCl, 5% (v/v) glycerol, and 1 mM PMSF, pH 8.0] and lysed by ultrasonification on ice. The cell debris and membranes were pelleted by centrifugation at 15,000 rpm (R20A2 rotor, Hitachi high-speed refrigerated centrifuge R21GIII) for 45 min at 4°C. The soluble C-terminally hexahistidine-tagged PaHigA was purified by affinity chromatography with nickel-nitrilotriacetic acid resin (Bio-Rad). Untagged proteins were removed with buffer A containing 50 mM imidazole. Recombinant PaHigA was then eluted with buffer A containing 250 mM imidazole. The protein was further purified by gel filtration (Superdex 200, GE Healthcare) equilibrated in buffer B [20 mM Tris, 300 mM NaCl, 5% (v/v) glycerol, pH 8.0] using an ÄKTA Purifier System (Amersham).
4
Recombinant Protein Expression Protocol
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KOD-plus DNA polymerase was purchased from Toyobo (Osaka, Japan). Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad (Hercules, CA, USA). RNase A inhibitor was purchased from Takara (Shiga, Japan). Oligodeoxyribonucleotides and oligoribonucleotides (Table S1 ) were synthesized by Invitrogen (Carlsbad, CA, USA) and Takara (Shiga, Japan), respectively. The expression vectors of pDEST17 (Invitrogen) and pET28-sumo were used throughout this study. E. coli strain DH5α was used in the gene cloning and Rosetta 2(DE3)pLysS (Novagen) strain was used to express recombinant protein. All other chemicals and reagents were of analytic grade.
5
Purification of P. furiosus Protein
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RNase A inhibitor was purchased from Takara (Dalian, China). KOD-plus DNA polymerase was purchased from Toyobo (Shanghai, China). Nickel-nitrilotriacetic acid resin was purchased from Bio-Rad (Shanghai, China). Oligodeoxyribonucleotides and oligoribonucleotides (Supplementary Table S1 ) were synthesized by Invitrogen (Shanghai, China) and Takara (Dalian, China), respectively. Expression vectors pDEST17 and pCDFDuet-1 were used throughout this study. Escherichia coli strain DH5α was used for cloning and Rosetta 2(DE3)pLysS strain was used to express P. furiosus protein. Mononucleotide CMP was purchased from Sigma. All other chemicals and reagents were of analytical grade.
6
Spacer Arm Incorporation in Oligonucleotides
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The spacer phosphoramidites dSpacer (dS), C3, C6, C12, S9, and S18 were purchased from Glen Research (Sterling, VA, USA) or ChemGenes Company (Wilmington, MA, USA) and were used to introduce the specific spacer arm into an oligonucleotide by solid–phase synthesis. The S. acidocaldarius strain was a gift from Professor Albers (Molecular Biology of Archaea, Institute of Biology, University of Freiburg, Freiburg, Germany), and its genomic DNA was extracted by phenol–chloroform. The expression vector pDEST17 and expression host Escherichia coli Rosetta2 (DE3) pLysS were used throughout this study. KOD-plus DNA polymerase was purchased from Toyobo (Osaka, Japan). Nickel-nitrilotriacetic acid resin was purchased from Bio–Rad (Hercules, CA, USA). All other chemicals and reagents were of analytical grade.
7
Recombinant Protein Expression in E. coli
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E. coli strain DH5α was used for gene cloning, and the Rosetta2(DE3)pLysS strain was used for recombinant protein expression. The expression vector pET28a was used to express the recombinant proteins. P. yayanosii genomic DNA was extracted from P. yayanosii cultures in our lab. PrimSTAR Max DNA polymerase was purchased from Takara (Shiga, Japan) . Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad (Hercules, CA, USA). Adenosine 3′,5′-bisphosphate (pAp) was purchased from Sigma (St Louis, MO, USA). The Cycle Pure Kit was purchased from Omega (Guangzhou, China). Methanol, formic acid, and ammonium formate were purchased from Aladdin (Shanghai, China) for the HPLC assay.
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