Ni ida

Manufactured by Macherey-Nagel

Sourced in United States

The Ni-IDA is a nickel-based immobilized metal affinity chromatography (IMAC) resin produced by Macherey-Nagel. It is designed for the purification of histidine-tagged recombinant proteins. The resin utilizes the strong interaction between nickel ions and the histidine residues present in the target proteins, allowing for efficient capture and separation.

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Lab products found in correlation

Bradford assay, by Bio-Rad (5 mentions) Pyruvate kinase, by Merck Group (3 mentions) Superdex s200, by Cytiva (3 mentions) Casein, by Merck Group (2 mentions) Malate dehydrogenase, by Merck Group (2 mentions) Ppiczα vector, by Thermo Fisher Scientific (1 mentions) Superdex 75 hiload 16 60 gel filtration column, by GE Healthcare (1 mentions) Fitc casein, by Merck Group (1 mentions) Superdex s200, by GE Healthcare (1 mentions) α casein, by Merck Group (1 mentions)

Spelling variants of this product

Protino Ni-IDA column (6 citations) Protino Ni-IDA Resin (5 citations) Protino Ni-IDA (4 citations) Ni-IDA 2000 Packed Columns Protino (2 citations) Protino 96 Ni-IDA plate (2 citations) Protino® Ni-IDA Packed Columns (2 citations)

7 protocols using ni ida

Heterologous Expression of CsVI1 in Pichia pastoris

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For the generation of Pichia pastoris expression plasmid, restriction sites KpnI and ApaI (NEB, Beijing, China) were used to clone the coding region of CsVI1 into the pPICZα vector (Invitrogen, Carlsbad, CA, USA). The ligation product was then transformed into E. coli competent DH5α cells by electroporation. Subsequently, the transformed bacterial cells were plated on low-salt LB medium supplemented with zeocin as a selection marker. Positive colonies were then used for vector amplification. The pPICZα plasmid (see above) carrying CsVI1 (and empty vector as a control) was linearized using PmeI, and then transformed into Pichia pastoris strain X-33 via electroporation. Further selection and protein purification were performed as previously described [37 (link)]. In short, after induction of the expression of CsVI1 recombinant protein in baffled Erlenmeyer flasks, the supernatant was precipitated with 80% ammonium sulfate. The protein pellets were then re-dissolved and dialyzed overnight. His6-tagged recombinant proteins were subsequently purified with 500 mg Ni-IDA (Macherey-Nagel, Allentown, PA, USA) according to the manufacturer’s instructions. Finally, the recombinant protein was eluted from the column with elution buffer.

Feng Z., Zheng F., Wu S., Li R., Li Y., Zhong J, & Zhao H. (2021). Functional Characterization of a Cucumber (Cucumis sativus L.) Vacuolar Invertase, CsVI1, Involved in Hexose Accumulation and Response to Low Temperature Stress. International Journal of Molecular Sciences, 22(17), 9365.

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Purification and Characterization of Ssb Protein

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Ssb or Ssb-GFP was purified from E. coli cells encoding Ssb N-terminally fused with a cleavable His₆-SUMO tag. Cells were lysed in buffer A (40 mM Hepes KOH pH 7.4, 150 mM KCl, 5 mM MgCl₂, 5% glycerol, 1 mM PMSF, 10 mM β-mercaptoethanol, 1 mM ETDA, DNaseI) and cleared lysates were mixed with nickel–iminodiacetic acid (Ni-IDA, Protino, Macherey-Nagel). After extensive washing with buffer A, His₆-SUMO-Ssb was eluted with buffer A containing 250 mM Imidazole. His₆-SUMO was removed by treatment with His₆-ULP protease during overnight dialysis against buffer A. His₆-ULP, His₆-SUMO and uncleaved His₆-SUMO-Ssb were removed by passing the eluate through a second Ni-IDA column. Ssb was loaded onto a HiLoad 16/60 Superdex 75 gel filtration column (GE Healthcare Life Sciences) and gel filtration was performed in buffer B (40 mM Hepes KOH pH 7.4, 50 mM KCl, 5 mM MgCl₂, 5% glycerol, 10 mM β-mercaptoethanol) containing 5 mM ATP. Aliquots were stored at - 80 °C.

Döring K., Ahmed N., Riemer T., Suresh H.G., Vainshtein Y., Habich M., Riemer J., Mayer M.P., O'Brien E.P., Kramer G, & Bukau B. (2017). Profiling Ssb-nascent chain interactions reveals principles of Hsp70 assisted folding. Cell, 170(2), 298-311.e20.

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Purification of E. coli ClpB and Chaperones

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E. coli strains used were derivatives of MC4100. ClpB was amplified by polymerase chain reaction (PCR), inserted into pDS56, and verified by sequencing. Mutant derivatives of clpB were generated by PCR mutagenesis and standard cloning techniques in pDS56 and were verified by sequencing. ClpB was purified after overproduction from E. coli ΔclpB::kan cells. ClpB wild-type and mutant variants were purified using Ni-IDA (Macherey-Nagel), following standard protocols, and size exclusion chromatography (Superdex S200) in MDH buffer [50 mM tris (pH 7.5), 150 mM KCl, 20 mM MgCl₂, 2 mM dithiothreitol (DTT)] supplemented with 5% (v/v) glycerol.
Purifications of DnaK, DnaJ, GrpE, and ClpP were performed as described previously (20 (link)). Pyruvate kinase of rabbit muscle, malate dehydrogenase of pig heart muscle, casein, and fluorescein isothiocyanate (FITC)–casein were purchased from Sigma. Protein concentrations were determined with the Bio-Rad Bradford assay.

Deville C., Carroni M., Franke K.B., Topf M., Bukau B., Mogk A, & Saibil H.R. (2017). Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase. Science Advances, 3(8), e1701726.

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Expression and Purification of ClpB and Associated Chaperones

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ClpB was amplified by PCR and inserted into pDS56 and verified by sequencing. Mutant derivatives of clpB were generated by PCR mutagenesis and standard cloning techniques in pDS56 and were verified by sequencing. ClpB was purified after overproduction from E. coli ΔclpB::kan cells. ClpB wild-type and mutant variants were purified using Ni-IDA (Macherey-Nagel) and size exclusion chromatography (Superdex S200, Amersham) following standard protocols. Purifications of DnaK, DnaJ, GrpE, Luciferase and Casein-YFP were performed as described previously (Haslberger et al., 2008 (link), Oguchi et al., 2012 (link), Seyffer et al., 2012 (link)). Pyruvate kinase of rabbit muscle and Malate Dehydrogenase of pig heart muscle were purchased from Sigma. Protein concentrations were determined with the Bio-Rad Bradford assay.

Deville C., Franke K., Mogk A., Bukau B, & Saibil H.R. (2019). Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor. Cell Reports, 27(12), 3433-3446.e4.

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Purification and Characterization of ClpC, MecA, and ClpP

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E. coli strains used were derivatives of MC4100, XL1-blue or DH5α. ClpC, MecA, ClpP were amplified by PCR, inserted into pDS56 and verified by sequencing. Mutant derivatives of ClpC were generated by PCR mutagenesis and standard cloning techniques in pDS56 and were verified by sequencing. Transformation into B. subtilis 168 was performed by standard methods (Anagnostopoulos and Spizizen, 1961 (link)). AmyE insertion in B. subtilis was checked by plating on agar containing 0.4% starch (w/v) additionally to appropriate antibiotics, screening for successful loss of α-amylase by staining starch with Lugol’s iodine.
ClpC and variants, MecA and ClpP were purified after overproduction from E. coliΔclpB::kan cells. GFP-SsrA was purified after overproduction from E. coli ΔclpX ΔclpP cells. All proteins were purified using Ni-IDA (Macherey-Nagel) and size exclusion chromatography (Superdex S200, GE Healthcare) following standard protocols. Pyruvate kinase of rabbit muscle, casein and FITC-casein were purchased from Sigma. Protein concentrations were determined with the Bio-Rad Bradford assay.

Carroni M., Franke K.B., Maurer M., Jäger J., Hantke I., Gloge F., Linder D., Gremer S., Turgay K., Bukau B, & Mogk A. (2017). Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control. eLife, 6, e30120.

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Purification and Characterization of ClpB

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E. coli strains used were derivatives of MC4100. ClpB was amplified by PCR and inserted into pDS56 and verified by sequencing. Mutant derivatives of clpB were generated by PCR mutagenesis and standard cloning techniques in pDS56 and were verified by sequencing. ClpB was purified after overproduction from E. coli ΔclpB::kan cells. ClpB wild type and mutant variants were purified using Ni-IDA (Macherey-Nagel) and size exclusion chromatography (Superdex S200, Amersham) following standard protocols. Purifications of DnaK, DnaJ, GrpE, Luciferase, and Casein-YFP were performed as described previously (Haslberger et al., 2008 (link); Oguchi et al., 2012 (link); Seyffer et al., 2012 (link)). Pyruvate kinase of rabbit muscle and Malate Dehydrogenase of pig heart muscle were purchased from Sigma. Protein concentrations were determined with the Bio-Rad Bradford assay.

Franke K.B., Bukau B, & Mogk A. (2017). Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase. Frontiers in Molecular Biosciences, 4, 6.

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Purification of E. coli Chaperone Proteins

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E. coli strains used were derivatives of MC4100. Mutant derivatives of ClpB/BAP were generated by PCR mutagenesis and standard cloning techniques in pDS56 and were verified by sequencing. Wild type and mutant ClpB were purified using Ni-IDA (Macherey–Nagel) and size exclusion chromatography (Superdex S200, Amersham) following standard protocols. Purifications of DnaK, DnaJ, GrpE, ClpP, Luciferase and Casein-YFP were performed as described previously (Oguchi et al., 2012 (link)). Pyruvate kinase and α–casein were purchased from Sigma. Protein concentrations were determined with the Bio-Rad Bradford assay.

Carroni M., Kummer E., Oguchi Y., Wendler P., Clare D.K., Sinning I., Kopp J., Mogk A., Bukau B, & Saibil H.R. (2014). Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation. eLife, 3, e02481.

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