Hispur ni nta

Manufactured by Thermo Fisher Scientific

Sourced in United States

The HisPur Ni-NTA is a chromatography resin designed for the purification of histidine-tagged recombinant proteins. It utilizes the high affinity interaction between nickel ions (Ni2+) and the histidine tag to selectively capture and purify the target protein.

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Lab products found in correlation

Centrifugal ultrafiltration devices, by Merck Group (2 mentions) Transfection enhancers, by Thermo Fisher Scientific (2 mentions) Opti mem, by Thermo Fisher Scientific (2 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions) Superdex 200, by Cytiva (2 mentions) Bio rex 70, by Bio-Rad (1 mentions) Cy5 maleimide monoreactive dye, by GE Healthcare (1 mentions) Monoq 4.6 100 pe, by GE Healthcare (1 mentions) Freestyle 293 f system, by Thermo Fisher Scientific (1 mentions) Mabselect sure, by Cytiva (1 mentions) Captureselect kappaxl, by Thermo Fisher Scientific (1 mentions) Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions) B per, by Thermo Fisher Scientific (1 mentions) Gibson assembly master mix, by New England Biolabs (1 mentions) Freestyle max reagent, by Thermo Fisher Scientific (1 mentions) Pet46 eklic vector, by Merck Group (1 mentions) Hiload 16 60 superdex 200 column, by GE Healthcare (1 mentions) Magnetic bead, by Thermo Fisher Scientific (1 mentions) Wizard2 automatic γ counter, by PerkinElmer (1 mentions) Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)

22 protocols using hispur ni nta

Purification and Labeling of Histones

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Expression and purification of Xenopus laevis core histone H2A, H2B, H3, and H4 and subsequent assembly of a histone octamer were performed following published protocols.^{39 (link)} H1G101C, a mutant of X. laevis linker histone H1° (termed H1 in this paper), was expressed and purified according to a previously published method.⁴⁰ After purification with a cation exchange column (Bio-Rex 70, 50–100 mesh, Bio-Rad Laboratories Inc., product no. 142-5832), H1G101C was labeled with the Cy5 maleimide monoreactive dye (GE Healthcare, product no. PA25031) according to the instructions recommended by the manufacturer. The labeling reaction mixture was purified with another cation exchange column (Bio-Rex 70, 100–200 mesh, Bio-Rad Laboratories Inc., product no. 142-5842) to remove any free dye. The labeling efficiency was close to 100% according to the absorbance values at 280 and 650 nm utilizing a correction factor of 0.05 for the dye at 280 nm. N-Terminally His₆-tagged yeast Nap1 (termed Nap1 in this paper) was overexpressed and purified according to previously published protocols.^{41 (link)} Briefly, yNap1 was purified with a Ni-NTA column (HisPur Ni-NTA, Thermo Scientific, product no. 88222) and a Mono-Q anion exchange column (Mono Q 4.6/100 PE, GE Healthcare, product code 17-5179-01).

Yue H., Fang H., Wei S., Hayes J.J, & Lee T.H. (2016). Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome. Biochemistry, 55(14), 2069-2077.

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Large-scale Protein Production Workflow

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Plasmids (500 ng DNA per ml culture) and polyethylenimine (MW 25,000; Polysciences; 3 μg per ml culture) were mixed with OptiMEM (Gibco; 100 μl per ml culture), incubated 20 minutes at room temperature and added to Expi293F cells at a density of 2 × 10⁶ / ml. Transfection Enhancers (ThermoFisher) were added 18–23 h post-transfection. Culture supernatant was harvested 4–6 days later by two centrifugation steps (800 × g for 10 minutes to remove cells and 20,000 × g for 20 minutes to remove debris). IgG1 Fc fused and 8his-tagged proteins were subsequently purified as previously described (27 ) using KANEKA KanCapA 3G Affinity (Pall) and HisPur Ni-NTA (Thermo Scientific) resins, respectively. Eluted proteins from affinity chromatography were then separated on a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) equilibrated with Dulbecco’s phosphate-buffered saline (PBS). Proteins from peak fractions were concentrated using centrifugal ultrafiltration devices (Millipore) to final concentrations of ~1 mg/ml (RBD-8h proteins), ~10 mg/ml (sACE2₂-8h proteins) and ~50 mg/ml (sACE2₂-IgG1 proteins). Concentrations were determined by absorbance at 280 nm using calculated extinction coefficients. Reported concentrations for sACE2₂ are based on monomeric subunits. Aliquots were snap frozen in liquid N₂ and stored at −80 °C.

Chan K.K., Tan T.J., Narayanan K.K, & Procko E. (2020). An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants. bioRxiv.

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Expression and Purification of CD19-HSA Fusion

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Antibodies and Fab were expressed in CHO cells (44 (link)). Capture from cell culture media was accomplished by affinity chromatography using either MabSelect SuRE (Cytiva Life Sciences) for antibodies or CaptureSelect KappaXL (Thermo Fisher Scientific) for Fab. Subsequent purification was done by size exclusion chromatography using Superdex 200 for antibodies and Superdex 75 for Fab (Cytiva Life Sciences).
Human CD19 ECD (amino acids 21–289) was fused to the N-terminus of C-terminally His-tagged human serum albumin (HSA) and expressed in the FreeStyle 293-F system (Thermo Fisher Scientific). Protein was captured from cell culture media by HisPur Ni-NTA (Thermo Fisher Scientific) and further purified by Superdex 200 (Cytiva Life Sciences). Purified CD19-HSA-His was biotinylated with EZ-Link Sulfo-NHS-LC biotin (Thermo Fisher Scientific).

Boyles J.S., Sadowski D., Potter S., Vukojicic A., Parker J., Chang W.Y., Ma Y.L., Chambers M.G., Nelson J., Barmettler B., Smith E.M., Kersjes K., Himes E.R., Lin C., Lucchesi J., Brahmbhatt J., Sina R., Martin J.A., Maestri E., Wiethoff C.M., Dyas G.L., Linnik M.D., Na S., Witcher D.R., Budelsky A, & Rubtsova K. (2023). A nondepleting anti-CD19 antibody impairs B cell function and inhibits autoimmune diseases. JCI Insight, 8(13), e166137.

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Purification of PTP Catalytic Domains

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All PTP catalytic domains and His₆-tagged constructs were expressed and purified using HisPur Ni-NTA (Thermo Scientific) according to the manufacturer’s instructions and as previously described for other His₆-tagged PTPs.^{18 (link)} After purification, proteins were exchanged into storage buffer (50 mM 3,3-dimethyl glutarate at pH 7.0, 1 mM EDTA, 1 mM TCEP), concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C. Bradford assays were used to measure enzyme concentrations and SDS-PAGE was used to assess purity.

Korntner S., Pomorski A., Krężel A, & Bishop A.C. (2018). Optimized allosteric inhibition of engineered protein tyrosine phosphatases with an expanded palette of biarsenical small molecules. Bioorganic & medicinal chemistry, 26(9), 2610-2620.

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Purification of Fluorescent Protein PAmCherry

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PAmCherry was purified from E. coli as previously described.^{[50 ]} Briefly, cells of E. coli DH5α containing the plasmid pBAD/HisB-PAmCherry1 (a gift from Vladislav Verkhusha [Addgene plasmid #31931]^{[6 (link)]}) were grown to mid-log phase, and protein expression was induced with 0.2% arabinose. Twenty hours after induction, cells were harvested by centrifugation and lysed using B-PER (ThermoScientific). His-tagged PAmCherry was purified using a spin column (HisPur™ Ni-NTA, ThermoScientific).

Tuson H.H., Aliaj A., Brandes E.R., Simmons L.A, & Biteen J.S. (2016). Addressing the Requirements of High Sensitivity Single-Molecule Imaging of Low-Copy Number Proteins in Bacteria. Chemphyschem : a European journal of chemical physics and physical chemistry, 17(10), 1435-1440.

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Production and Purification of 501Y.V2 RBD

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501Y.V2 RBD (encoding amino acid mutations K417N, E484K, and N501Y, and a C-terminal His-tag) was synthesized (Integrated DNA Technologies), and cloned into a mammalian expression vector (pcDNA3.1), using a Gibson Assembly Mastermix (New England Biolabs). Spike ectodomain (prefusion stabilized with 6 prolines³⁶ (link)) and RBD were produced by the transient transfection of Freestyle 293-F cells using FreeStyle MAX reagent (Thermo Fisher) or polyethylenimine (PEI), respectively. The HIS-tagged Spike ectodomain and RBD were purified from filtered supernatant using nickel IMAC resin (HisPur Ni-NTA, Thermo Fisher Scientific) followed by size-exclusion chromatography on a Superdex 200 (Cytiva) in PBS. On the day of immunization, indicated doses were mixed with 50 μg Matrix-M adjuvant (Novavax AB, Uppsala, Sweden) in a final inoculation volume of 800 μl.
Macaques were immunized intramuscularly (i.m.) with half of each dose administered in each quadricep. All immunizations and blood samplings were performed under sedation with 10-15 mg/kg ketamine (Ketaminol, Intervet, Sweden) administered i.m.. Blood plasma was isolated by centrifugation, and heat inactivated at 56°C for 60 min.

Sheward D.J., Mandolesi M., Urgard E., Kim C., Hanke L., Perez Vidakovics L., Pankow A., Smith N.L., Castro Dopico X., McInerney G.M., Coquet J.M., Karlsson Hedestam G.B, & Murrell B. (2021). Beta RBD boost broadens antibody-mediated protection against SARS-CoV-2 variants in animal models. Cell Reports Medicine, 2(11), 100450.

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Purification and Characterization of Recombinant Human Neuraminidases

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Human recombinant neuraminidase 2, NEU3, and NEU4 were expressed as N‐terminal maltose binding protein fusion proteins in Escherichia coli and purified as previously reported.¹² Because production of active recombinant NEU1 requires mammalian cells, the human enzyme was expressed as a His‐tagged protein in HEK293 cells, transduced with a CathA‐IRES‐NEU1 lentivirus,¹³ and partially purified by affinity chromatography using HisPur Ni‐NTA (Thermo Fisher Scientific; 88222). For lectin blotting experiments, commercially available purified recombinant human NEU1 (NEU1‐156H; Creative Biomart) was used. Neuraminidase activity and inhibition assays were performed using 2′‐(4‐methylumbelliferyl)‐α‐d‐N‐acetylneuraminic acid and GM3 ganglioside as substrates, as described.¹⁴

Demina E.P., Smutova V., Pan X., Fougerat A., Guo T., Zou C., Chakraberty R., Snarr B.D., Shiao T.C., Roy R., Orekhov A.N., Miyagi T., Laffargue M., Sheppard D.C., Cairo C.W, & Pshezhetsky A.V. (2021). Neuraminidases 1 and 3 Trigger Atherosclerosis by Desialylating Low‐Density Lipoproteins and Increasing Their Uptake by Macrophages. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 10(4), e018756.

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Purification of Recombinant β-Catenin

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A construct comprising residues 126–686 of human β-catenin (AB451264) was sub-cloned into pET-46 Ek/LIC vector (EMD Millipore). The recombinant His-fusion β-catenin was expressed in E. coli BL21 (DE3) and purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (HisPur™ Ni-NTA, Thermo Scientific). The protein was eluted from the resin with 250 mM imidazole, 150 mM NaCl, 20 mM Tris, pH 8.0, and purified further on a gel-filtration HiLoad 16/60 Superdex-200 column (GE Healthcare) equilibrated with buffer (150 mM NaCl, 20 mM CAPS, pH 10.5).

Shin S.H., Lim D.Y., Reddy K., Malakhova M., Liu F., Wang T., Song M., Chen H., Bae K.B., Ryu J., Liu K., Lee M.H., Bode A.M, & Dong Z. (2017). A Small Molecule Inhibitor of the β-Catenin-TCF4 Interaction Suppresses Colorectal Cancer Growth In Vitro and In Vivo. EBioMedicine, 25, 22-31.

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Purification of His-tagged Protein Tyrosine Phosphatases

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His₆-tagged PTPs (SHP-1cat, SHP-1fl, and FAP-1cat) were expressed and purified using HisPur Ni-NTA (Thermo Scientific) according to the manufacturer’s instructions and as described [22 (link)], with one change: IPTG (0.5 mM) inductions were carried out at 18°C for 20 hours. After purification, proteins were exchanged into storage buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 25 mM CaCl₂, 1 mM TCEP), concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C. Bradford assays and SDS-PAGE were used to measure enzyme concentrations.

, & Bishop A.C. (2016). A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases. Biochemical and biophysical research communications, 481(1-2), 153-158.

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Recombinant Protein Purification with Intein

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The DNA sequences encoding the 2x-HHTC-Re protein constructs containing an intein self-cleavage site, hexahistidine and Lumio ® detection tags, were commercially synthesized (IDT, gBlocks®) and were assembled into the PSB1C3 iGEM backbone using Gibson Assembly (NEB), transformed NEB DH5a® competent E. coli for plasmid construction and transformations plated on chloramphenicol selective LB plates. The transformations were incubated at 37 °C overnight and DNA constructs confirmed in select colonies using verification primers and colony PCR. Plasmids from sequence-verified clones were then transformed into the T7 Express® E. coli (NEB) protein production strain. His-tag purification on crude cell extract from our colonies using ThermoFisher HisPur® Ni-NTA spin columns. The standard protein purification protocol was modified by introducing a buffer containing 50 mM DTT for on-site cleavage, so the desired fusion protein could be eluted. We then performed a BCA Assay to determine our total protein concentration in each elution. Following protein de-salting with Amcon® filter spin columns, we confirmed the presence of target protein in the final elution using ThermoFisher Scientific Lumio® Tag Detection Kit.

Urbina J., Patil A., Fujishima K., Paulino-Lima I.G., Saltikov C, & Rothschild L.J. (2019). A new approach to biomining: Bioengineering surfaces for metal recovery from aqueous solutions. Scientific Reports, 9, 16422.

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