Ni2 nta column

Manufactured by GE Healthcare

Sourced in United States

The Ni2+-NTA column is a type of affinity chromatography column used for the purification of histidine-tagged proteins. It utilizes the high affinity interaction between the nickel ions (Ni2+) immobilized on the column's resin and the histidine tags present on the target proteins, allowing for their selective capture and separation from a complex mixture.

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Lab products found in correlation

Fastpfu dna polymerase, by Transgene (2 mentions) Pd 10 desalting column, by GE Healthcare (2 mentions) Ni2 nta, by Qiagen (2 mentions) Superdex 200 16 60 column, by GE Healthcare (2 mentions) Microplate reader, by Agilent Technologies (1 mentions) E colibl21 de3 cells, by Takara Bio (1 mentions) Bradford protein assay kit, by Bio-Rad (1 mentions) Red cell lysis buffer, by Tiangen Biotech (1 mentions) His tag, by Beyotime (1 mentions) Protein g coupled agarose beads, by Merck Group (1 mentions) Protease inhibitor cocktail, by Selleck Chemicals (1 mentions) Rapamycin, by Selleck Chemicals (1 mentions) Bafilomycin a1, by Selleck Chemicals (1 mentions) Beclin 1, by Cell Signaling Technology (1 mentions) Rapidstep ecl reagent, by Merck Group (1 mentions) Antibodies against gapdh, by Beyotime (1 mentions) Clodronate liposome, by Liposoma (1 mentions) Pd miditrap g 25, by GE Healthcare (1 mentions) Superdex g200 column, by GE Healthcare (1 mentions) Source 15q column, by GE Healthcare (1 mentions)

22 protocols using ni2 nta column

Purification and Crystallization of PBP2 Mutant

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The constructs encoding PBP2-6140CT with A501 mutations comprise residues 44–581 of PBP2 fused in-frame to the C-terminal end of His₆-tagged maltose-binding protein (MBP) with an intervening tobacco etch virus (TEV) protease site. The protein was expressed at 18°C, the cells lysed, and the fusion protein was purified from the soluble fraction on a Ni²⁺-NTA column (GE Healthcare, Piscataway, NJ). The peak fractions were pooled and dialyzed at 4 °C at a 10:1 molar ratio of MBP-PBP2 to His₆-TEV, which resulted in nearly complete digestion of the fusion protein. The mutant PBP2 variants were then rerun over a Ni²⁺-NTA column to remove His₆-MBP, uncleaved fusion protein, and His₆-TEV. The purified proteins were concentrated to ~20 mg ml⁻¹ in 20 mM Tris•HCl, pH 8.0, 500 mM NaCl, 10% glycerol and stored at −80°C until ready for use.
Crystals of PBP2-6140CT-A501T were grown by vapor diffusion at 24°C in hanging drops, where the reservoir contained 2.2 M ammonium sulfate buffered with 100 mM Tris pH 8.25. Crystals were cryo-protected by passage through mother liquor to which glycerol had been added to a final concentration of 30% (v/v), followed by flash freezing in liquid nitrogen.

Tomberg J., Fedarovich A., Vincent L.R., Jerse A.E., Unemo M., Davies C, & Nicholas R.A. (2017). Alanine-501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness. Biochemistry, 56(8), 1140-1150.

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Recombinant Protein Purification from E. coli

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Following cultivation of E. coli BL21 Star (DE3) cells harboring the desired constructs, the induction and purification of target proteins were performed as described previously (14 (link)). The His-tagged target proteins were subjected to purification through a Ni²⁺-NTA column (GE Healthcare, Chicago, IL, USA). The fractions containing rEDIII-CaM and VP1 peptide-TrxA proteins were confirmed by 12% polyacrylamide gel containing 1% sodium dodecyl sulfate (12% SDS-PAGE). The rEDIII protein, which was expressed from pET21d-rEDIII (14 (link)), was produced and purified as described previously (14 (link), 25 (link)).

Yang M.H., Hu C.C., Wong C.H., Liang J.J., Ko H.Y., He M.H., Lin Y.L., Lin N.S, & Hsu Y.H. (2021). Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling. Frontiers in Immunology, 12, 739837.

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Recombinant Enzyme Purification and Assay

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Enzymes
were expressed in E. coliBL21 (DE3) cells (Takara, Dalian, China) using pET derived
plasmids. The recombinant cells were first grown in the LB medium
supplemented with 50 μg/mL kanamycin at 37 °C until the
OD600 reached 0.6 and gene expression was induced by adding 0.1 mM
isopropyl β-d-thiogalactopyranoside (IPTG) for an additional
18 h at 20 °C. The harvested cells were washed twice with 20
mM Tris-HCl buffer (pH 7.0) and suspended in a buffer of 50 mM Na₂HPO₄ (pH 7.0), 0.2 mM EDTA, and 0.1 mM dithiothreitol.
Suspended cells were disrupted by sonication and centrifuged at 100 000g for 1 h. The supernatant was purified using a Ni²⁺-NTA column (GE Healthcare Bio-Sciences, Piscataway, NJ) to obtain
samples for the activity assay. The purity of enzymes was checked
by SDS-PAGE (Bio-Rad Laboratories, Hercules) and the protein concentrations
were quantified using a Bradford protein assay kit (Bio-Rad Laboratories,
Hercules).
The in vitro activity of TD was
measured following the previous reports. The reaction of each activity
assay contained 20 μM PLP, 50 mM potassium phosphate buffer
(pH 7.5), 10 mM threonine, an appropriate amount of the enzyme, and
varied concentrations of isoleucine. The reaction was carried out
at 30 °C and the formation of α-ketobutyrate was measured
at 230 nm using a BioTek Microplate Reader. Each measurement has been
repeated three times.

Wu M., Sun Y., Zhu M., Zhu L., Lü J, & Geng F. (2021). Molecular Dynamics-Based Allosteric Prediction Method to Design Key Residues in Threonine Dehydrogenase for Amino-Acid Production. ACS Omega, 6(16), 10975-10983.

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Autophagy Signaling Pathway Analysis

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Antibodies against LC3 I/II, p70S6 K, ULK, and beclin 1 were purchased from Cell Signaling Technology. Antibodies against GAPDH and His-Tag were purchased from Beyotime. Antibodies against TLR2 and TLR4 were purchased from Abcam. Antibody against p-mTOR was purchased from Santa Cruz. Protein-G coupled agarose beads and RapidStep™ ECL Reagent were purchased from Millipore. Ni²⁺-NTA column was purchased from GE lifesciences. DMEM High glucose culture medium was purchased from Hyclone. Cell lysis buffer for Western and IP and Nitric Oxide Synthase Assay Kit was purchased from Beyotime. Protease Inhibitor Cocktail is purchased from Bimake. Red Cell Lysis Buffer was purchase from Tiangen. Rapamycin and Bafilomycin A1 were purchased from Selleck. Clodronate Liposome was purchased from Liposoma.

Shu Z., Yuan J., Wang H., Zhang J., Li S., Zhang H., Liu Y., Yin Y, & Zhang X. (2020). Streptococcus pneumoniae PepO promotes host anti-infection defense via autophagy in a Toll-like receptor 2/4 dependent manner. Virulence, 11(1), 270-282.

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Purification of SNARE Complex Proteins

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syt1, cpx2, EIIA^Glc, MSP1D1, and SNAREs were expressed in BL21 STAR^TM (DE3) and purified using GSTrap and Ni²⁺-NTA columns^{20 (link),25 (link),44 (link)–47 (link)}. To produce spMSP1D1,spNW15, spNW25, spNW30, spNW50, spNW80, and spNW100, plasmids were transformed into BL21 STAR^TM (DE3) cells that were grown in LB supplemented with Km (50 mg/ml) to OD₆₀₀ ~ 0.7. Protein expression was induced with 0.2 mM IPTG at 16 °C overnight. Bacteria were harvested by centrifugation at 3450 × g for 20 min, resuspended in buffer A (50 mM Tris-HCl (pH 8),100 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), and lysed on ice using a Branson cell disrupter (60% duty cycle, 45 secs). Cell lysates were clarified by centrifugation at 12,000 × g for 45 mins. The supernatants were loaded onto a 1 ml Ni²⁺-NTA column (GE Healthcare), followed by extensive wash (20 column volume) using buffer B (50 mM Tris-HCl (pH 8), 20 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol). Proteins were eluted in buffer C (50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), desalted in buffer A using PD MiDiTrap G-25 (GE Healthcare), and stored at −80 °C.

Zhang S., Ren Q., Novick S.J., Strutzenberg T.S., Griffin P.R, & Bao H. (2021). One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 12, 5451.

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Cloning and Purification of RdAcuH and PrpE

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The RdacuH gene was amplified from the genome of Roseovarius nubinhibens ISM by PCR using FastPfu DNA polymerase. PCR primers were designed with the NdeI and XhoI restriction sites. The amplified RdacuH is recombined to the vector pET-22b. FastPfu DNA polymerase and the vector pET-22b were purchased from TransGen Biotech (China) and Novagen company (Germany), respectively.
All site-directed mutations were introduced using overlap PCR and verified by sequencing. The constructed recombinant plasmids, including the wild type RdacuH and its mutants were transferred into E. coli BL21 (DE3) for expression. The cells were cultured in LB medium with 0.1 mg/ml ampicillin at 37°C to an OD₆₀₀ of 1.0-1.2. Then the culture was induced at 20°C overnight with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Wild type RdAcuH and its mutants were purified by affinity chromatography on a Ni²⁺-NTA column (GE healthcare, United States), and then fractionated by anion exchange chromatography on a Source 15Q column (GE healthcare, United States) and gel filtration on a Superdex G200 column (GE healthcare, United States).
The prpE gene was amplified from the genomic DNA of Dinoroseobacter shibae DFL 12. Expression and purification of PrpE were conducted with the same methods as RdAcuH described above.

Cao H.Y., Wang P., Xu F., Li P.Y., Xie B.B., Qin Q.L., Zhang Y.Z., Li C.Y, & Chen X.L. (2017). Molecular Insight into the Acryloyl-CoA Hydration by AcuH for Acrylate Detoxification in Dimethylsulfoniopropionate-Catabolizing Bacteria. Frontiers in Microbiology, 8, 2034.

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Expression and Purification of Tle1 and VgrG/Hcp

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Primers were designed according to the sequences of the tle1^AH and vgrG genes of A. hydrophila NJ-35 in GenBank (accession number NZ_CP006870). Tle1^AH was cloned into the pGEX-4T-1 vector (Invitrogen) for expression with an N-terminal glutathione-S-transferase (GST) tag. VgrG/hcp was cloned into the pET-28a vector (Invitrogen) with a His tag. The GST-Tle1^AH and His-VgrG/Hcp proteins were expressed in BL21 (DE3) cells (CWBIO, Beijing, China). The transformed cells were cultured in LB medium at 37 °C to an OD₆₀₀ of 0.8, at which time the fusion protein expression was at the highest level and most was expressed as soluble protein not as an inclusion body based on our preliminary experiment. Protein expression was induced with 100 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 °C for 20 h. The cultures were harvested by centrifugation at 8000 × g, resuspended in 1 × PBS and lysed by sonication. The lysate was centrifuged at 12 000 × g for 15 min at 4 °C to remove precipitate, and then the supernatant was loaded on a Ni²⁺-NTA column (GE Healthcare, Shanghai, China) to purify the proteins. The eluted proteins were collected and dialyzed for pull-down assays.

Ma S., Dong Y., Wang N., Liu J., Lu C, & Liu Y. (2020). Identification of a new effector-immunity pair of Aeromonas hydrophila type VI secretion system. Veterinary Research, 51, 71.

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Recombinant Basigin Protein Production

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A section of the basigin gene encoding immunoglobin domains 1 and 2 of the short isoform (residues 22-205) was amplified from cDNA using primers: 5′-CCGGATCCGCTGCCGGAACCGTGTTC-3′ and CCCATATGCTAGTGTGACCGCACTCTCAGG-3′. PCR products were cloned into a modified pET15b vector (Novagen), which encodes an N-terminal hexa-histidine tag followed by a tobacco etch virus (TEV) protease cleavage site. TEV cleavage leaves an additional glycine at the N-terminus from the cleavage site.
Basigin was expressed in bacterial strain Origami B (DE3) (Novagen) by incubation overnight at 25°C after induction with 1mM IPTG. The protein was purified by nickel-nitrilotriacetic acid (Ni²⁺-NTA; Qiagen) affinity chromatography, followed by buffer exchange into PBS using a PD-10 desalting column (GE Healthcare), and overnight cleavage with His-tagged TEV protease at 4°C, before a second Ni²⁺-NTA column. The flow-through was concentrated using an Amicon Ultra centrifugal filter device (molecular mass cutoff, 3,000 Da). Finally, gel filtration was performed with a Superdex 200 16/60 column (GE Healthcare) in 20 mM HEPES (pH 7.5) and 150 mM NaCl.

Wright K.E., Hjerrild K.A., Bartlett J., Douglas A.D., Jin J., Brown R.E., Illingworth J.J., Ashfield R., Clemmensen S.B., de Jongh W.A., Draper S.J, & Higgins M.K. (2014). Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies. Nature, 515(7527), 427-430.

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E. coli Expression and Purification of DARPin9_29-HSP70

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Freshly transformed E. coli BL21 (DE3) cells were grown in SOB medium containing 0.1 g/L ampicillin; the lac-promoter was induced with 1 mM ITPG at OD₆₀₀ of 0.7. Expression was allowed to continue for 12 h at 37 °C. Cells were harvested by centrifugation at 6000× g for 15 min at 4 °C, and the cell pellet was resuspended in lysis buffer (20 mM Tris-base, 20 mM NaCl, 20 mM MgCl₂, and 1 mg/mL lysozyme). The suspension was incubated for 30 min at room temperature and then sonicated on ice. Cellular debris was removed by centrifugation at 15,000× g for 30 min at 4 °C.
The solution was applied to a Ni²⁺-NTA column (GE Healthcare) equilibrated in 100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, and pH 8.0 buffer (B1). DARPin9_29-HSP70 was eluted with pH 4.5 B1 buffer. The protein yield was 10 mg/L of growth medium.

Alekseeva L.G., Ovsyanikova O.V., Schulga A.A., Grechikhina M.V., Shustova O.A., Kovalenko E.I., Svirshchevskaya E.V., Deyev S.M, & Sapozhnikov A.M. (2024). Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair. Cells, 13(4), 317.

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Cloning and Purification of SBPs

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The genes encoding SBPs from R. pomeroyi DSS-3 or R. nubinhibens ISM were amplified from the genome of R. pomeroyi DSS-3 or R. nubinhibens ISM by PCR using FastPfu DNA polymerase (TransGen Biotech, China). The dmpX homologue from P. sp. strain HTCC7211 (locus tag HTCC7211_00013840) was synthesized by the Beijing Genomics Institute (China). The related genes were then cloned into the NdeI/XhoI restriction sites of the pET-22b vector (Novagen, Germany) with a C-terminal His tag. Point mutations in DmpX were introduced using the PCR-based method and verified by DNA sequencing. The SBPs and corresponding mutant forms were produced in E. coli BL21 (DE3). The cells were cultured in the LB medium with 0.1 mg/ml ampicillin at 37 °C to an OD₆₀₀ of 0.8–1.0 and then induced at 18 °C for 16 h with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After induction, cells were collected by centrifugation, resuspended in the lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5% glycerol, pH 8.0), and lysed by a pressure crusher. The proteins were first purified by affinity chromatography on a Ni²⁺-NTA column (GE Healthcare, US), and then fractionated by gel filtration on a Superdex G75 column (GE healthcare, US).

Li C.Y., Mausz M.A., Murphy A., Zhang N., Chen X.L., Wang S.Y., Gao C., Aguilo-Ferretjans M.M., Silvano E., Lidbury I.D., Fu H.H., Todd J.D., Chen Y, & Zhang Y.Z. (2023). Ubiquitous occurrence of a dimethylsulfoniopropionate ABC transporter in abundant marine bacteria. The ISME Journal, 17(4), 579-587.

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