Ni2 affinity chromatography

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Ni2+-affinity chromatography is a laboratory technique used for the purification and separation of proteins and biomolecules. It utilizes the specific interaction between nickel ions (Ni2+) immobilized on a solid support and histidine-tagged or polyhistidine-tagged proteins, allowing for the selective capture and isolation of the target molecules from complex mixtures.

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Lab products found in correlation

Mwco 10 000, by Merck Group (1 mentions) Superdex 200 10 30 gl column, by GE Healthcare (1 mentions) Kta purifier, by GE Healthcare (1 mentions) Bl21 codonplus ril, by Agilent Technologies (1 mentions) Rosetta 2, by Merck Group (1 mentions) Quikchange protocol, by Agilent Technologies (1 mentions) Amicon ultra 15, by Merck Group (1 mentions) Q sepharose chromatography, by GE Healthcare (1 mentions) Bradford protein assay, by Bio-Rad (1 mentions) Superdex 200 26 60 column, by GE Healthcare (1 mentions) Positively charged nylon membrane, by Merck Group (1 mentions) Streptavidin hrp, by Beyotime (1 mentions) Heparin hp column, by GE Healthcare (1 mentions) Akta fplc system, by GE Healthcare (1 mentions) Bca assay kit, by Tiangen Biotech (1 mentions)

10 protocols using ni2 affinity chromatography

Phototropin LOV1 from C. reinhardtii Purification

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Phototropin-LOV1-C57S (CrLOV1) from the unicellular algae C. reinhardtii was expressed from a plasmid carrying an N-terminal 10x His-tag.^{28 (link)} For UV/vis and EPR spectroscopy, CrLOV1 was expressed in Escherichia coli strain BL21 (DE3) using LB-medium. The protein for¹⁵N
photo-CIDNP experiments was expressed in M9 minimal medium with ¹⁵N isotope-enriched ¹⁵NH₄Cl.^{17 (link)} Both types are purified by Ni²⁺-affinity
chromatography (kta purifier, GE Healthcare). Residual imidazole was
removed by dialysis against phosphate buffer (300 mM NaCl and 50 mM
KH₂PO₄/K₂HPO₄, pH 8.0).
About 20 mg of protein was concentrated using an ultracentrifugation
filter with a molecular cutoff at 10 kDa (Merck Millipore, MWCO 10000).
The appropriate volume of trehalose solution (1.2 M stock solution)
was added to the concentrated protein sample achieving a molar ratio
of 50:1 (trehalose to protein). The mixture of trehalose and CrLOV1 was dried on a Petri dish under nitrogen gas for
several hours until a solid amorphous glass was formed.

Kurle-Tucholski P., Wiebeler C., Köhler L., Qin R., Zhao Z., Šimėnas M., Pöppl A, & Matysik J. (2024). Red Shift in the Absorption Spectrum of Phototropin LOV1 upon the Formation of a Semiquinone Radical: Reconstructing the Orbital Architecture. The Journal of Physical Chemistry. B, 128(18), 4344-4353.

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Purification of Truncated PARN Proteins

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The plasmid containing the cDNA sequence of the wild type human PARN was kindly provided by Professor Anders Virtanen (Uppsala University, Sweden). The truncated proteins p60 (residues 1–520 AA) and p46 (residues 1–446 AA) were constructed by standard protocols of mutagenesis using the following primers: p60-forward, 5′-CGATGTCACATATGGAGATAATCAGGAGC-3'; p60-reverse 5′-GATCCTCGAGCTACTTCTCTTCCTGTTTTC-3'; p46-forward, 5′-GCTACTCGAGCTTCTCTTCCTGTTTTC-3'; and p46-reverse, 5′-GATCGTCGACTTAATGATCACGTTTAGGCTGC-3'. The obtained genes were cloned to the expression vector pET-28a (Novagen) and verified by sequencing. The recombinant proteins were overexpressed in Escherichia coli BL21 (DE3) (Stratagene, Heidelberg, Germany) and purified as described previously [18 (link),28 (link)]. In brief, the expression of the proteins was induced by 0.1 mM IPTG at 16 °C for 24 h. The proteins were isolated from the supernatant fraction of cell lysates by Ni²⁺-affinity chromatography (GE Healthcare),. The final products were purified using a Superdex 200 10/30 GL column equipped on an ÄKTA purifier (GE Healthcare). The protein concentration was determined according to the Bradford method [29 (link)]. The protein solutions used for analysis were prepared in buffer A containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM DTT, 0.2 mM EDTA and 20% (v/v) glycerol.

He G.J, & Yan Y.B. (2019). Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association. Biochemistry and Biophysics Reports, 18, 100626.

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AurA Kinase Domain Mutagenesis Protocol

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Site-directed mutagenesis of AurA kinase domain (122–403) in a pET30-based vector was carried out using a QuikChange protocol (Agilent). The mutations were C290A, C393A, and either F275C for the 275 construct or T288C and T287A for the 288 construct. Proteins were expressed in E. coli BL21-CodonPlus -RIL (Agilent) or Rosetta 2 (Merck) DE3 cells in LB medium, with initial growth at 37°C followed by overnight incubation at 21°C after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside. Bacterial pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 10% glycerol, 40 mM imidazole), supplemented with protease inhibitors and DNase (Roche), lysed by sonication and clarified by centrifugation. Lysates were filtered then subjected to Ni²⁺ affinity chromatography (GE Healthcare). Proteins were eluted in lysis buffer containing 250 mM imidazole, purified to homogeneity by S75 size exclusion chromatography (GE Healthcare) (gel filtration buffer: 50 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl₂, 10% glycerol, 10 mM β-mercaptoethanol (BME)), then concentrated to ∼10 mg/mL and flash frozen for future use. Protein concentrations were measured in triplicate with a ND-1000 Spectrophotometer (NanoDrop) using molecular weights and extinction coefficients calculated by ProtParam (ExPASy).

Rowan F., Richards M., Widya M., Bayliss R, & Blagg J. (2014). Diverse Functionalization of Aurora-A Kinase at Specified Surface and Buried Sites by Native Chemical Modification. PLoS ONE, 9(8), e103935.

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Recombinant Atlantic Cod Parvalbumin Production

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rGad m 1 (recombinant Gad m 1) was produced from a pET15b construct containing the synthetic ORF of Atlantic cod parvalbumin A5187436 . The mutants were prepared using Quick-Change protocols and a pair of complementary oligonucleotides (Supplementary Table S2). The proteins were produced in BL21(DE3) cells and purified from the soluble fraction by Ni²⁺-affinity chromatography (GE Healthcare Life Sciences), followed by Q-Sepharose chromatography (GE Healthcare Life Sciences)36 . The eluted fractions were filtered using 30 kDa-pore size Amicon Ultra-15 (Merck Millipore) and extensively dialyzed against 5 mM Hepes, pH 7.5, containing 0.1 mM CaCl₂36 . Before use, the proteins were centrifuged at 16,000 × g for 20 min at 4 °C to remove any insoluble material. Protein concentrations were determined using the Bradford protein assay (Bio-Rad) calibrated with BSA (Sigma, A8806). Aβ42 was obtained from GenScript and used as previously described57 (link).

Sánchez R., Martínez J., Castro A., Pedrosa M., Quirce S., Rodríguez-Pérez R, & Gasset M. (2016). The amyloid fold of Gad m 1 epitopes governs IgE binding. Scientific Reports, 6, 32801.

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Purification of MST3 Kinase Domain

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The kinase domain was purified by FPLC using Ni²⁺-affinity chromatography (GE Healthcare) in 50 mM Tris (pH 8), 300 mM NaCl, and a gradient of 10–300 mm imidazole. The eluted fusion protein was digested overnight with PreScission protease at 4 °C, followed by a second Ni²⁺-affinity column. The flowthrough was then loaded onto a Superdex200 26/60 column (GE Healthcare) in 50 mm Tris (pH 8), 150 mm NaCl, 1 mm DTT. The resulting eluate yielded crystallization-grade monomeric enzyme. The full-length protein was purified using a similar approach with a few changes. The Ni²⁺-affinity columns were run using 50 mm Tris (pH 8), 150 mm NaCl, and a gradient of 10–250 mm imidazole. In addition, to remove PreScission from the solution, the protein was subjected to a glutathione column in 50 mm Tris (pH 8), 150 mm NaCl, 1 mm DTT, and a gradient of 0–10 mm reduced glutathione. The flow-through from this column was then loaded onto the Superdex200 26/60 in the same buffer as above. Purified MST3 KD was concentrated to 25 mgmL⁻¹ for crystallization studies, and both KD and FL proteins were concentrated to 10 mgmL⁻¹ for DSF studies and stored at −80°C.

Olesen S.H., Zhu J.Y., Martin M.P, & Schönbrunn E. (2016). Discovery of Diverse Small-Molecule Inhibitors of Mammalian Sterile20-like Kinase 3 (MST3). ChemMedChem, 11(11), 1137-1144.

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Cloning and Mutational Analysis of H4

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Human H4 cDNA (NM_175054.2) was cloned from HeLa cells using the following primers: 5′-GATATCGATGTCTGGGCGAGGTAAAGGTGG-3’ (forward) and 5′-CGGGATCCTCAACCGCCGAAACCATAAA-3’ (reverse). Full-length H4 was cloned into pFlag-CMV4 plasmid. The H4 (R3Q) mutant was generated using the QuickChange Site-directed Mutagenesis Kit and the following primers: 5′-CCTTGCCACCTTTACCTTGCCCAGACATCGATATC-3' (forward) and 5′-GATATCGATGTCTGGGCAAGGTAAAGGTGGCAAGG-3' (reverse). Recombinant proteins FEN1, Pol β, APE1, PCNA were expressed in Escherichia coli BL21DE and were purified using Ni²⁺ affinity chromatography (GE Healthcare). DNA Ligase I (Lig I) was purchased from Abnova.

Ma Z., Wang W., Wang S., Zhao X., Ma Y., Wu C., Hu Z., He L., Pan F, & Guo Z. (2020). Symmetrical dimethylation of H4R3: A bridge linking DNA damage and repair upon oxidative stress. Redox Biology, 37, 101653.

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Recombinant TaASR1-D Protein Purification

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The ORF of TaASR1‐D was cloned into pET‐28a(+), and then, the recombinant vector was transformed into E. Coli Rosetta (DE3) competent cell (CWBIO, Beijing, China). The histidine‐tagged TaASR1‐D‐recombinant protein was purified using Ni²⁺ affinity chromatography (GE Healthcare, Waukesha, USA), and the probes labelled with or without biotin were synthesized by TSINGKE Biological Technology Co., Ltd. (Wuhan, China). The EMSA was performed according to the method described with modifications (Yu et al.,2019 ). In brief, the binding buffer contained 10 mM Tris‐HCl pH 7.5, 2 mM MgCl₂, 50 μM ZnCl₂, 50 mM NaCl, 0.2 mM EDTA and 10% (v/v) glycerol, and samples were separated on a 10% native polyacrylamide gel in 0.5 × TBZ buffer (45 mM Tris‐H₃BO₃ pH 8.0, 1 mM ZnCl₂). Then, the samples were transferred to a positively charged nylon membrane (Millipore, Bedford, MA, USA), and the detection was performed using HRP‐Streptavidin at a 1:4000 dilution (Beyotime, Shanghai, China).

Qiu D., Hu W., Zhou Y., Xiao J., Hu R., Wei Q., Zhang Y., Feng J., Sun F., Sun J., Yang G, & He G. (2021). TaASR1‐D confers abiotic stress resistance by affecting ROS accumulation and ABA signalling in transgenic wheat. Plant Biotechnology Journal, 19(8), 1588-1601.

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Purification and Characterization of SaPriA

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The gene SAAV1184, encoding SaPriA, was amplified by PCR using genomic DNA of Staphylococcus aureus subsp. aureus ED98 as template. The forward (5′-GGGAAGGATCCATGATAGCGAAAGTCA-3′) and the reverse (5′-CCATTCTCGAGCATCATCATCTGTGGA-3′) primers were designed, and BamHI and XhoI restriction sites into SaPriA were introduced. The PCR product was then ligated to the pET21b vector. SaPriA was purified by Ni²⁺-affinity chromatography (GE Healthcare Bio-Sciences) eluted with Buffer A (20 mM Tris–HCl, 250 mM imidazole, and 0.5 M NaCl, pH 7.9). After dialysis against Buffer B (20 mM HEPES and 100 mM NaCl, pH 7.0), the SaPriA solution was further purified by the Heparin HP column (GE Healthcare Bio-Sciences), eluted with a linear NaCl gradient from 0.1 to 1.0 M with Buffer B using the AKTA-FPLC system (GE Healthcare Bio-Sciences). SDS-PAGE was used to show the protein purity (Fig. 1). By the vanadate-sensitive colorimetric assay, the ATPase activity of SaPriA was detected: more the concentration of inorganic phosphate released by ATP hydrolysis, more the OD₆₁₀ intensity. The binding of kaempferol to SaPriA was analyzed by the fluorescence emission spectra of SaPriA quenched by kaempferol.Fig. 1

SDS-PAGE of the purified SaPriA and molecular mass standards

Huang Y.H., Huang C.C., Chen C.C., Yang K.J, & Huang C.Y. (2015). Inhibition of Staphylococcus aureus PriA Helicase by Flavonol Kaempferol. The Protein Journal, 34(3), 169-172.

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Purification of Recombinant E2 Protein

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The HEK293-APPV-E2 cell line was cultured for eight days and the supernatant of cells was harvested and filtered through a 0.45-μm pore-sized membrane. The recombinant E2 protein was purified through Ni2⁺ affinity chromatography according to the manufacturer’s guidelines (GE Healthcare, USA). The purified protein was separated with 12.5% SDS-PAGE and observed after Coomassie brilliant blue staining. The protein concentration was determined using a BCA assay kit according to the manufacturer’s manual (Tiangen, Beijing, China).

Song H., Gao X., Li J., Dong X., Fu Y., Shao L., Zhang J., Qiu H.J, & Luo Y. (2024). Development and application of an indirect ELISA for detection of antibodies against emerging atypical porcine pestivirus. Virology Journal, 21, 53.

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Histidine-Tagged Protein Purification

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The histidine tag was used to facilitate the purification of the target protein. The recombinant cells were cultivated in 2-L flasks, which contained 100 mL of LB medium for 36 h, and harvested by centrifugation at 6,000 × g for 20 min. The resuspended cells were disrupted with a Scientz-II D ultrasonic homogenizer (Scientz Biotechnology, Ningbo, China). The crude CEase obtained from the previous step was filtered and purified by Ni 2+ affinity chromatography from GE Healthcare (Uppsala, Sweden). All purification steps were implemented according to the manufacturer's protocol (Instruction 71-5001-87 AE, http:// wolfson .huji .ac .il/ purification/ PDF/ Tag _Protein _Purification/ Ni -NTA/ AMERSHAM _Chelating _Instruction .pdf; GE Healthcare) at 4°C. The purified protein was dialyzed against 50 mM sodium phosphate buffer (pH 7.0) for 8 h.

Chen Q., He W., Yan X., Zhang T., Jiang B., Stressler T., Fischer L, & Mu W. (2018). Construction of an enzymatic route using a food-grade recombinant Bacillus subtilis for the production and purification of epilactose from lactose. Journal of dairy science, 101(3).

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