DH5a cells were transformed with PET30a plasmids, and protein was induced with 0.4 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 16°C for 12 to 16 h. nsp14 was purified with nickel-nitrilotriacetic acid (Ni-NTA) resin (GenScript) as described previously (19 (link), 24 (link)).
Ni nta resin
Manufactured by GenScript
Sourced in China
Ni-NTA resin is a solid support material used for the purification of recombinant proteins containing a histidine (His) tag. The resin is composed of nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. The His-tagged proteins bind to the Ni-NTA functional groups on the resin, allowing for selective capture and purification from complex mixtures.
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Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Yuanye Bio-Technology (1 mentions)
Gel extraction kit, by Promega (1 mentions)
Tetrahydrofolate thf, by Merck Group (1 mentions)
Fastdigest restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Dna extraction kit, by Promega (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Genjet plasmid preparation kit, by Thermo Fisher Scientific (1 mentions)
Luria bertani medium, by HiMedia (1 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
10 kda cutoff concentrator, by Merck Group (1 mentions)
Arvo luminometer, by PerkinElmer (1 mentions)
Glutathione resin, by GenScript (1 mentions)
Protease inhibitor cocktail, by Targetmol (1 mentions)
Tre mre, by MedChemExpress (1 mentions)
Purkine mbp tag dextrin resin 6ff, by Abbkine (1 mentions)
Typhoon trio imager, by GE Healthcare (1 mentions)
14 protocols using ni nta resin
1
Purification of nsp14 Protein from DH5a Cells
2
Cloning and Purification of Recombinant Enzymes
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All chemicals used in this study were of analytical grade or higher quality. Pyridoxal 5‐phosphate monohydrate (PLP), dithiothreitol (DTT), NADH, and NAD+ were purchased from Yuanye Bio‐Technology (Shanghai, China). β‐Mercaptoethanol (β‐ME) and lipoic acid were obtained from Aladdin (Shanghai, China). Tetrahydrofolate (THF) was obtained from Sigma‐Aldrich (St. Louis, MO, USA). Phusion high‐fidelity DNA polymerase and FastDigest restriction enzymes were purchased from Thermo‐Fisher Scientific (Pittsburgh, PA, USA). T4 DNA ligase was purchased from New England Biolabs (Ipswich, MA, USA). DNA extraction kit and gel extraction kit were purchased from Promega (Madison, WI, USA). The pET‐22b(+) and pET28a(+) vectors were purchased from Novagen (Darmstadt, Germany). All synthetic oligonucleotides were ordered from GeneWiz (Suzhou, China). Competent cells of E. coli TOP10 and E. coli BL21 (DE3) were purchased from WEIDI Ltd. (Shanghai, China). Ni‐NTA resin was purchased from Genscript (Nanjing, China). BCA protein assay kit was purchased from SolarBio (Beijing, China).
3
Recombinant Protein Expression in E. coli
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E. coli strains TOP10, BL21(DE3) and BL21(DE3)pLysS, and pET-28a and pET-32a plasmids were obtained from Novagen (Madison, WI). KpnI, XhoI, and NdeI restriction enzymes, T4 DNA Ligase, GeneJet Gel Extraction kit, and GenJet plasmid Preparation kit were obtained from Thermo scientific (MD, USA). Luria-Bertani (LB) medium was purchased from HiMedia (Mumbai, Maharashtra, India). Ni-NTA resin was provided by GenScript (Piscataway, USA). All recombinant DNA manipulation procedures were performed according to standard molecular biology techniques.[19 ]
4
Purification and Characterization of scFv16
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The codon-optimized nucleotide sequence of scFv16 was synthesized by GenScript and subcloned into an expression vector pFastBac1 with an 8x His tag at the C-terminus. The scFv16 used in this paper was the same as that used in the structures of the CB1-Gi-scFv1635 (link). In brief, scFv16 was expressed in secreted form from Trichuplusia ni Hi5 insect cells and purified by Ni-NTA chromatography. The supernatant was incubated with Ni-NTA resin (GenScript) at 4 °C for 2 h. The resin was then loaded to a gravity column and washed with 15 column volumes (CV) of wash I buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 10 mM imidazole; followed by 15 CV of wash II buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 30 mM imidazole. The protein was eluted with elute buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 250 mM imidazole. The elute was collected and further purified using a Superdex 200 10/300 column (GE Healthcare). Monomeric fractions were pooled, concentrated 10 mg/mL with a 10-kDa cut-off concentrator (Millipore), and flash frozen in liquid nitrogen, then stored at −80 °C for further use.
5
Purification and Kinase Assay of YL2-His
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To obtain purified YL2-His protein, full-length YL2 cDNA was amplified using the primers YL2-HIS-F and YL2-HIS-R (Supplementary Table 2) then subcloned into the vector pET44a. The resulting construct and empty vector were expressed in Escherichia coli strain BL21. Recombinant YL2-His was purified using Ni-NTA resin (GenScript) and used for kinase assay. The UMP kinase assay was performed as described previously (Yoshida et al. 2012 (link)). The 100 μL assay mixture consisted of 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 0.2 mM UMP, and 10 μM ATP. The reaction was started by the addition of various amounts of purified His-tagged YL2 protein and incubated at 30 °C for 0.5 h. Next, 100 μL of the Kinase-Glo Assay reagent was added to initiate the luciferase reaction. The luminescence intensity was measured after 10 min incubation at room temperature using an ARVO luminometer (PerkinElmer, America).
6
Purification of His-tagged SlyA Protein
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His6-tagged SlyA was expressed in the E. coli strain BL21 (DE3) harboring the appropriate plasmid. Briefly, 15 mL of the overnight culture was added in 300 mL LB broth and grown until the OD600 reached 0.6–0.8. Upon the addition of IPTG to a final concentration of 0.2 mM, bacterial culture was incubated further in a shaker at 18°C for 16–18 h. Bacterial cells (from 300 mL of culture) were lysed in 30 mL of ice cold PBS buffer via sonication and cell lysates were clarified by centrifugation at 5,000 ×g for 10 min three times at 4°C. His-tagged proteins were captured with Ni-NTA Resin (GenScript) and washed serially with PBS and 20 mM imidazole. Finally, proteins were eluted with 300 mM imidazole. Purified proteins were further dialyzed overnight in 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% (vol/vol) glycerol, and 1 mM DTT with at least two buffer changes.
7
Purification and Chitin Binding Assay of Recombinant PbChia1
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The full-length cDNA of PbChia1 without signal peptide was amplified by PCR using PbChia1-BamHI-F and PbChia1-BamHI-R primers (Table S3) with BamHI restriction enzyme sites and inserted into pGEX-6P1 and pET-28a, for GST fusion and His-tagged recombinant protein, respectively. GST fusion proteins and His-tagged proteins were purified using glutathione resin (GenScript, Cat. No. L00206) and Ni-NTA resin (GenScript, Cat. No. L00250), respectively, and then stored at −80 °C. After purification of the fusion proteins, the chitin pull-down assay was performed as previously described [48 (link)].
8
Purification of TRE/MRE-Gαs-Gβ-Gγ Complexes
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For purification, cells were thawed in 20 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM MgCl2, and CaCl2 with Protease Inhibitor Cocktail (TargetMol). For TRE/MRE-IP-Gαs-Gβ-Gγ complexes, 10 μM TRE/MRE (MedChemExpress) and 2 mg of Nb35 were added and incubated at room temperature for 1 hour. Membranes were solubilized with 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG; Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS; Anatrace) at 4°C for 2 hours and then centrifuged at 70,000g for 30 min to remove insoluble material. Solubilized complexes were immobilized on nitrilotriacetic acid (Ni-NTA) resin (GenScript), washed with 30 column volumes of wash buffer, and eluted with 300 mM imidazole. Complexes were concentrated using 100-kDa Amicon filters and further purified by size exclusion chromatography on a Superdex 6 Increase 10/300 GL column (GE Healthcare) pre-equilibrated with size buffer containing 20 mM Hepes (pH 7.4),150 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) glyco-diosgenin [(GDN) Anatrace], 0.00025% digitonin (w/v), 0.00015% CHS, and 10 μM ligand to separate complexes. Eluted fractions were analyzed by SDS–polyacrylamide gel electrophoresis, and those containing receptor-Gs complexes were pooled, concentrated, and used for cryo-EM.
9
Recombinant Protein Purification and REMSA
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The recombinant protein was created with a fusion of MBP in the PPR75641–756 N-terminus and 6xHis in the C-terminus, and was purified across two columns equipped with Ni2+ affinity resin (Ni-NTA Resin, GenScript) and MBP (PurKine MBP-Tag Dextrin Resin 6FF, Abbkine) in turn. The control fusion protein containing only MBP and His tags was purified as well. RNA probes (Supplementary Table S1 ) were synthesized and labeled with 6-FAM at the 5′ end by GenScript (Nanjing, China). For the RNA electrophoresis mobility shift assays (REMSAs), the dialyzed recombinant protein was incubated with 100 fmol RNA probes in a 10 × binding buffer (100 mM HEPES PH = 7.3, 200 mM KCl, 10 mM MgCl2, 10 mM DTT) condition and reacted in a 20 μl system for 30 min at 25°C with 10 units of RNasin, followed by separation on 8% native acrylamide gels in a 0.5× TBE buffer. After electrophoresis, the gels were screened using a Typhoon Trio Imager (GE).
10
Csy Complex-AcrIF14 Interaction Assay
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His-tagged Csy complex and wild-type or mutant AcrIF14 proteins were purified according to the procedure described above. 8 μM His-tagged Csy complex was first incubated with Ni-NTA resin (GenScript) in 20 μl buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl and 5% glycerol at 18°C for 10 min, and then 40 μM AcrIF14 or its mutants were added in the system and incubated at 37°C for 30 min. The resins were washed three times with buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl and 50 mM imidazole. The resins were then analyzed through 8–16% SDS–PAGE with Coomassie blue staining (CBS).
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