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Ni sepharose resin

Manufactured by Cytiva
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Ni Sepharose resin is a nickel-charged affinity chromatography media used for the purification of histidine-tagged proteins. It provides a simple and efficient method for the capture and purification of recombinant proteins expressed with a histidine tag.

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Lab products found in correlation

E coli rosetta cells, by Merck Group (2 mentions) Source 15q column, by Cytiva (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Superdex 75 10 300 gl column, by Cytiva (1 mentions) Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions) Superose 6 10 300 column, by Cytiva (1 mentions) Immobilon psq pvdf membrane, by Merck Group (1 mentions) Mouse anti his, by Thermo Fisher Scientific (1 mentions) Goat anti mouse igm hrp, by Thermo Fisher Scientific (1 mentions) Pierce ecl 2 western blotting substrate, by Thermo Fisher Scientific (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Superdex 75, by Cytiva (1 mentions) Nupage 4 to 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions) Ncoi hf, by New England Biolabs (1 mentions)

Close spelling variants of this product

Ni Sepharose excel resin Ni Sepharose affinity resin Ni Sepharose 6 Fast Flow resin Ni Sepharose High Performance resin Ni-Sepharose HP resin Ni Sepharose

7 protocols using ni sepharose resin

1

Purification and Biotinylation of Fusion Proteins

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GFP, SUMO, and GFP and SUMO fused with the extracellular domain of CD99 (ECD; residues 23-122 of UniProt ID P14209) containing a 6xHis tag, AviTag, and TEV cleavage site at the N-Terminal region were cloned into pHBT plasmids 72 (link). BL21(DE3) cells were transformed, separately, with plasmids. The target proteins were expressed via isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 18°C for 20 h. The targets were purified via immobilized metal affinity chromatography using a Ni-Sepharose resin (Cytivia). The purified proteins were biotinylated in vitro using in-house prepared recombinant BirA enzyme and further purified by size-exclusion chromatography using a Superdex 75 10/300 GL column (Cytivia).
Romero L.A., Hattori T., Ali M.A., Ketavarapu G., Koide A., Park C.Y, & Koide S. (2021). High-valency anti-CD99 antibodies toward the treatment of T cell acute lymphoblastic leukemia. Journal of molecular biology, 434(5), 167402.
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2

Production and Purification of Stabilized SARS-CoV-2 Spike Proteins

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Soluble 6P-stabilized SARS-CoV-2 spike proteins (WT, BA.1, BA.2, BA.3 and BA.4/5) were expressed by transient transfection20 (link),36 (link). In brief, the genes encoding spike ECD of different variants were synthesized and codon-optimized by GenScript, and then cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). The plasmid was transfected using PEI into FreeStyle 293-F cells (Invitrogen). Transfected cells were cultured at 35 °C, 8% CO2, and the cell culture supernatant was collected following 4 to 5 days of incubations. Protein was purified from filtered cell supernatants using Ni Sepharose resin (Cytiva) and further purified by gel filtration chromatography using a Superose 6 10/300 column (Cytiva) in 1 × TBS (20 mM Tris-HCl, 200 mM NaCl, pH8.0).
Guo H., Yang Y., Zhao T., Lu Y., Gao Y., Li T., Xiao H., Chu X., Zheng L., Li W., Cheng H., Huang H., Liu Y., Lou Y., Nguyen H.C., Wu C., Chen Y., Yang H, & Ji X. (2023). Mechanism of a rabbit monoclonal antibody broadly neutralizing SARS-CoV-2 variants. Communications Biology, 6, 364.
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3

Truncation Analysis of CD99 Extracellular Domain

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Kunkel mutagenesis was utilized to make truncated CD99 ECD fragments (23-122, 33-122, and 43-122 ) and a combination of Kunkel mutagenesis and restriction and insertion cloning were utilized to produce the other CD99 fragments (53-122, 63-122, 73-122, 83-122, 93-122, 103-122, and 113-122) 77 (link). The proteins were expressed and purified as described above, except they were only purified using a Ni-Sepharose resin (Cytivia). SDS-PAGE of 50 ng of each purified protein was performed followed by transfer to Immobilon-PSQ PVDF membrane (ca# ISEQ00010, Millipore). For Western blot detection, the membranes were blocked with 5% skim milk overnight at 4°C, then washed three times with 1X TBST (50 mM Tris pH 7.5, 150 mM NaCl, and 0.05% Tween-20). The membranes were then incubated with 14 nM of clones 10, 22, and 30, 1 nM of HO36-1.1, or 1:1000 of mouse anti-His (ca# 37-2900, Invitrogen) for 1.5 hours at room temperature, then washed as described above. The membranes were then incubated with goat anti-mouse IgG Fc conjugated with horseradish peroxidase (HRP) (ca# 31437, Invitrogen) or goat anti-mouse IgM-HRP (ca# 626820, Invitrogen), for 1 hour at room temperature, then washed as described above. Pierce ECL 2 Western Blotting Substrate (ca# 80198, Thermo) was added according to manufacturer protocol. Signal detection was analyzed on ChemiDoc Touch imaging system (Biorad).
Romero L.A., Hattori T., Ali M.A., Ketavarapu G., Koide A., Park C.Y, & Koide S. (2021). High-valency anti-CD99 antibodies toward the treatment of T cell acute lymphoblastic leukemia. Journal of molecular biology, 434(5), 167402.
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4

Purification of DsBC-tagged Protein

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Cells were harvested and lysed by sonication in sonication buffer (20 mM Hepes pH 7.5, 400 mM NaCl, 5% glycerol). After centrifugation, the supernatant was applied to the Ni Sepharose resin (Cytiva). The DsBC and His6 tag were cleaved by adding HR3C protease to the resin and incubating for 16 h at 4 °C. Eluted protein was further purified with SEC (Superdex 75, Cytiva) in the SEC buffer (20 mM Hepes pH 7.5 and 400 mM NaCl).
Uchikawa E., Chen Z., Xiao G.Y., Zhang X, & Bai X.C. (2021). Structural basis of the activation of c-MET receptor. Nature Communications, 12, 4074.
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5

Conversion of Phage Clones to scAb Antibodies

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Plasmid DNA of peptide binding phage clones were pooled together and their single chain Fv (scFv) genes isolated by restriction digestion using NotI-HF and NcoI-HF (R3189S and R3193S, New England Biolabs, Ipswich, MA, USA) and cloned into the bacterial expression vector pIMS147 [41 (link)]. The ligated DNA was ethanol precipitated and used to transform electrocompetent TG1 cells (605021, Lucigen, Middleton, WI, USA). Individual colonies were selected, and DNA sequencing of plasmids confirmed successful conversion of all positive phage clones into the scAb format.
Selected scAb clones were grown in TB medium and induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for protein expression in bacterial periplasm [41 (link)]. Periplasmic extracts containing histidine-tagged scAbs were purified using immobilized metal affinity chromatography (IMAC) with Ni Sepharose resin (17371202, Cytiva, Marlborough, MA, USA). The concentration of purified scAbs was measured using Pierce™ BCA Protein Assay Kit (23225, Thermo Scientific, Waltham, MA, USA) as per manufacturer’s protocol. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed using NuPAGE™ 4 to 12%, Bis-Tris protein gel (NP0321PK2, Thermo Scientific, Waltham, MA, USA) to assess scAb purity.
Tan T.H., Patton E., Munro C.A., Corzo-Leon D.E., Porter A.J, & Palliyil S. (2021). Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein. Viruses, 13(11), 2201.
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6

Purification of DDX39B and Variants

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All proteins were expressed in E. coli Rosetta cells (Sigma-Aldrich). Protein expression was induced by 0.5 mM IPTG at 20°C overnight. Cells were lysed in a lysis buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, 1 mM PMSF, and 5 mg/L aprotinin. The GST-tagged proteins were pulled down using Glutathione Sepharose 4B resin and the His-tagged DDX39B was pulled down using Ni Sepharose resin (Cytiva). GST-tagged DDX39B-CTD variants were digested with GST-TEV overnight and purified on a Source 15Q column (Cytiva). The proteins were then passed over Glutathione Sepharose 4B resin to remove remaining GST and uncleaved protein. Purified DDX39B-CTD variants were concentrated, aliquoted, and stored at −80°C in 10 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, and 10% glycerol. The other affinity-purified proteins were first purified on a mono Q column and were subjected to overnight digestion with GST-TEV (for GST-tagged proteins) or His-TEV (for His-tagged DDX39B) to remove the affinity tag. The digested proteins were passed over Glutathione Sepharose 4B resin or Ni Sepharose resin to remove uncleaved protein and TEV. The proteins were further purified on a Superdex 200 column equilibrated with 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5 mM TCEP. All purified proteins were concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.
Xie Y., Gao S., Zhang K., Bhat P., Clarke B.P., Batten K., Mei M., Gazzara M., Shay J.W., Lynch K.W., Angelos A.E., Hill P.S., Ivey A.L., Fontoura B.M, & Ren Y. (2023). Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly. Cell reports, 42(8), 112988.
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7

Purification of DDX39B and Variants

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All proteins were expressed in E. coli Rosetta cells (Sigma-Aldrich). Protein expression was induced by 0.5 mM IPTG at 20°C overnight. Cells were lysed in a lysis buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, 1 mM PMSF, and 5 mg/L aprotinin. The GST-tagged proteins were pulled down using Glutathione Sepharose 4B resin and the His-tagged DDX39B was pulled down using Ni Sepharose resin (Cytiva). GST-tagged DDX39B-CTD variants were digested with GST-TEV overnight and purified on a Source 15Q column (Cytiva). The proteins were then passed over Glutathione Sepharose 4B resin to remove remaining GST and uncleaved protein. Purified DDX39B-CTD variants were concentrated, aliquoted, and stored at −80°C in 10 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, and 10% glycerol. The other affinity-purified proteins were first purified on a mono Q column and were subjected to overnight digestion with GST-TEV (for GST-tagged proteins) or His-TEV (for His-tagged DDX39B) to remove the affinity tag. The digested proteins were passed over Glutathione Sepharose 4B resin or Ni Sepharose resin to remove uncleaved protein and TEV. The proteins were further purified on a Superdex 200 column equilibrated with 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5 mM TCEP. All purified proteins were concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.
Xie Y., Gao S., Zhang K., Bhat P., Clarke B.P., Batten K., Mei M., Gazzara M., Shay J.W., Lynch K.W., Angelos A.E., Hill P.S., Ivey A.L., Fontoura B.M, & Ren Y. (2023). Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly. Cell reports, 42(8), 112988.
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