Chelating sepharose

Manufactured by GE Healthcare

Sourced in United Kingdom, Sweden

Chelating Sepharose is a chromatography resin used for the purification and separation of proteins, peptides, and other biomolecules. It consists of agarose beads with chelating groups that can selectively bind metal ions, enabling the capture and purification of target molecules.

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Nickel-activated chelating Sepharose column Nickel-complexed Chelating Sepharose Ni2+-chelating Sepharose Fast Flow column Chelating Sepharose Fast Flow column Chelating sepharose beads Ni2+ chelating Sepharose Ni2+-chelating Sepharose column Ni2+ charged Chelating Sepharose Fast Flow FF Chelating Sepharose resin Ni-chelating Sepharose affinity chromatography

21 protocols using chelating sepharose

Purification and Deuteration of P. furiosus PF0014 Protein

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P. furiosus PF0014 expressed with an N-terminal hexahistidine tag was purified using a Ni²⁺-immobilized affinity column (Chelating Sepharose, GE Healthcare) from E. coli soluble lysate and then directly subjected to complex formation with PbaA. To prepare deuterated proteins, bacterial cells were grown in an M9 minimal medium containing glucose as a mixture with 25% of isotopically natural form and 75% of fully deuterated from (1,2,3,4,5,6,6-D₇, 98%, Cambridge Isotope Laboratories, Inc.), along with 25% H₂O and 75% D₂O, as previously described^{23 (link)}. To determine PF0014 positions in the complex, Trx-PF0014 was used. Trx-PF0014 with an N-terminal hexahistidine tag was purified using a Ni²⁺-immobilized affinity column (Chelating Sepharose, GE Healthcare). After cleavage of the hexahistidine tag by thrombin protease, Trx-PF0014 was purified using a HiTrapQ HP anion exchange column (GE Healthcare) and HiLoad Superdex 200 column (GE Healthcare) in 20 mM Tris–HCl (pH 8.0) containing 200 mM NaCl and 2 mM dithiothreitol (DTT).

Yagi-Utsumi M., Sikdar A., Song C., Park J., Inoue R., Watanabe H., Burton-Smith R.N., Kozai T., Suzuki T., Kodama A., Ishii K., Yagi H., Satoh T., Uchiyama S., Uchihashi T., Joo K., Lee J., Sugiyama M., Murata K, & Kato K. (2020). Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation. Scientific Reports, 10, 1540.

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Purification of GluN1b-GluN2A ATD Complex

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The High Five cell culture medium containing secreted GluN1b-GluN2A ATD fusion proteins was collected, concentrated, and dialyzed against 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl using tangential flow filtration. The concentrated sample supplemented with 5 mM imidazole was load onto a Chelating Sepharose (GE Healthcare) conjugated with cobalt (Co-NTA), washed with 20 Column Volumes (CV) of 40 mM imidazole, 10 CV of 80 mM imidazole and eluted with 10 CV of 300 mM imidazole. The octa-histidine tags and 57-link were removed by thrombin digestion at 18°C for 20 h. The proteins were concurrently de-glycosylated by endoglycosidase, EndoF1, at a 1:1 (w:w) ratio of GluN1b-GluN2A ATD and EndoF1 in 20 mM Tris-HCl and 200 mM NaCl at pH 8. The digested samples were recharged onto the Cobalt-NTA at 20 mM imidazole and the flow-though was collected. At this step Fab29 is added to the protein at a 1:2.25 molar ratio (GluN1b-GluN2A ATD:Fab). The mixture was subjected to a size-exclusion chromatography (Superdex200) in the presence of 10 μM ZnCl₂ and the peak fraction containing the GluN1b-GluN2A ATD-Fab complex was collected. Expression and purification of GluN1b-GluN2B ATD was done as described previously described (Karakas et al., 2011 (link)) with the exception that there was no addition of ifenprodil or any other ligands during the purification process.

Romero-Hernandez A., Simorowski N., Karakas E, & Furukawa H. (2016). Molecular basis for subtype-specificity and high-affinity zinc inhibition in the GluN1-GluN2A NMDA receptor amino terminal domain. Neuron, 92(6), 1324-1336.

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Proteasome Subunit Interactions Probed by Pull-Down Assays

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The 3xFLAG-tagged α4, GST-fused proteasome α5-subunit (GST-α5), non-tagged or His₆-tagged forms of proteasome α1, α2, α3, α6, and α7 subunits, and scPAC3/4 were used in the pull-down assays. For immobilization, 20 μg of His₆-tagged scPAC3/4 or GST-α5 was applied to Ni²⁺-charged Chelating Sepharose or Glutathione Sepharose 4B (GE Healthcare) resins, respectively. The His₆-scPAC3/4-immobilized resins were incubated with 50 μg of α1–α7 subunits for 2 h at 4 °C in an incubation buffer (20 mM Tris-HCl (pH 8.0) and 150 mM NaCl). For α5, the GST-α5-immobilized resins were incubated with 50 μg of α1–α4, α6, and α7 in the presence and absence of 50 μg of scPAC3/4 as described above. Since α7 makes a stable complex with α6 [19 (link),20 (link)], the pull-down experiments containing α7 were performed separately. The resins were washed four times with the incubation buffer, which contains 60 mM imidazole in the His₆-tag pull-down assays. Proteins bound to the Chelating or Glutathione Sepharose resins were eluted using 20 mM Tris-HCl (pH 8.0)/500 mM imidazole or 50 mM Tris-HCl (pH 8.0)/10 mM reduced glutathione, respectively, and analyzed by SDS-PAGE, stained with CBB.

Satoh T., Yagi-Utsumi M., Okamoto K., Kurimoto E., Tanaka K, & Kato K. (2019). Molecular and Structural Basis of the Proteasome α Subunit Assembly Mechanism Mediated by the Proteasome-Assembling Chaperone PAC3-PAC4 Heterodimer. International Journal of Molecular Sciences, 20(9), 2231.

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Recombinant LAMP1 and mAb Production

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Nucleic acid sequences coding for LAMP1 extracellular domains fused to his-tag or Halotag-histag at the C-terminal or coding for the antibody heavy or light chains were cloned into mammalian expression plasmids under the CMV enhancer/promoter and the SV40 polyA signal. Resulting plasmids were transfected into HEK293 cells (Thermo Fisher Scientific; K9000-10) using FreeStyle^™ MAX 293 Expression System according to the manufacturer’s instructions. LAMP1 proteins were purified by immobilized metal affinity chromatography (Chelating Sepharose, 17-0575-01 GE Healthcare) and stored in PBS after concentration and buffer exchange (Sephadex G-25 column, GE Healthcare).
For initial characterization, mAbs were produced at 30 mL scale, purified by protein A affinity chromatography and stored in PBS after desalting on mini trap Sephadex G-25 column. For the developability study, mAbs were produced at 1 L scale and purified by a two-step process including protein A affinity chromatography (HiScreen MabSelect Sure protein A, GE Healthcare, 28-9269-77) and size-exclusion chromatography (HiLoad 26/600 superdex 200 pg, GE Healthcare, 28-9893-36).

Cameron B., Dabdoubi T., Berthou-Soulié L., Gagnaire M., Arnould I., Severac A., Soubrier F., Morales J., Leighton P.A., Harriman W., Ching K., Abdiche Y., Radošević K, & Bouquin T. (2020). Complementary epitopes and favorable developability of monoclonal anti-LAMP1 antibodies generated using two transgenic animal platforms. PLoS ONE, 15(7), e0235815.

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Fusion Proteins for Protein Expression

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Genes encoding the different fusion proteins were codon optimized for expression in Escherichia coli (E. coli), synthetized, and cloned into the pET-15b vector by Genewiz (NJ, US). We fused the NADH oxidase from Thermus thermophilus (NOX) with the LCD of Dbp1 from Saccharomyces cerevisiae (from residue 1 to 155), Laf1 from Caenorhabditis elegans (from residue 1 to 168), and Ddx4 from human (from residue 1 to 236). E. coli BL21-GOLD (DE3) competent cells were transformed with the corresponding plasmids encoding NOX, Ddx4-NOX, Dbp1-NOX, and Laf1-NOX, and grown aerobically in Luria-Bertani broth (LB) medium supplemented with 100 µg/mL ampicillin. Protein expression was induced at OD₆₀₀ of 0.6 with 0.5 mM isopropyl d-thiogalactopyranoside (99%, PanReac AppliChem) for 16 h at 20 ^oC.
NOX enzyme was purified using a heat shock method^{37 (link)}. Dbp1-NOX, Laf1-NOX, and Ddx4-NOX proteins were purified by a combination of heat shock and immobilized metal ion affinity chromatography (Chelating Sepharose, GE Healthcare) according to a standard protocol^{29 (link),30 (link)}. The final purity was confirmed by SDS-PAGE, and protein concentration was determined by measuring absorbance at 280 nm.

Gil-Garcia M., Benítez-Mateos A.I., Papp M., Stoffel F., Morelli C., Normak K., Makasewicz K., Faltova L., Paradisi F, & Arosio P. (2024). Local environment in biomolecular condensates modulates enzymatic activity across length scales. Nature Communications, 15, 3322.

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Recombinant Protein Production Using GB1 Tag

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Constructs containing a 20 amino acid region from the S1/S2 linker were fused via a linker with the sequence ELQGGGGG to the GB1 domain (immunoglobulin-binding domain of streptococcal protein G) and a C-terminal 6xHis tag. The pET28a vectors were transformed to E. coli BL21 cells and protein expression was induced at 37 °C by addition of 1 mM IPTG. The His-tagged proteins were purified by cobalt affinity chromatography using Chelating Sepharose (GE Healthcare). The proteins were eluted in buffer containing 25 mM Hepes–KOH (pH 7.4), 300 mM NaCl, 10% glycerol, 200 mM imidazole.

Örd M., Faustova I, & Loog M. (2020). The sequence at Spike S1/S2 site enables cleavage by furin and phospho-regulation in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV. Scientific Reports, 10, 16944.

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Monomeric scFv Protein Purification

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The scFv-AP fusion protein, described above, is a dimer as AP is a homodimer. To produce monomeric scFv for crystallization experiments, the D2 scFv was cloned into the pEB04 vector with Sfi-digestion. This construct generates scFv with a C-terminal His-tag. The scFv-6xHis protein was expressed in E. coli (AD494) and purified on a Chelating Sepharose (GE Healthcare, Ridgewood, NJ) column according to manufacturer’s instructions.

Goldgur Y., Susi P., Karelehto E., Sanmark H., Lamminmäki U., Oricchio E., Wendel H.G., Nikolov D.B, & Himanen J.P. (2014). Generation and characterization of a single-chain anti-EphA2 antibody. Growth factors (Chur, Switzerland), 32(6), 214-222.

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Purification of Recombinant Protein E5

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The E5 recombinant protein was purified from heat-treated extracts first by immobilized metal affinity chromatography (IMAC) using chelating Sepharose (GE Healthcare) charged with nickel (NiSO₄, 200 mM). Unbound proteins were washed away with PBS and bound proteins were eluted in a two-step gradient using PBS containing 10 and 250 mM imidazole, respectively. IMAC elution fractions containing the target protein E5 were desalted against 20 mM Tris (pH 7.5) and E5 was further purified by ion exchange chromatography (IEX) using a prepacked HiTrap Q FF column. The E5 protein was eluted using a three-step gradient (20 mM Tris pH 7.5 with 240, 500 and 1000 mM NaCl, respectively). The buffer was exchanged against PBS overnight at 4°C using a Spectra/Por membrane (MWCO 6000–8000 g/mol; SpectrumLabs). The samples were then concentrated using Vivaspin centrifugal concentrators (Sartorius-Stedim Biotech GmbH) and passed through a 0.22-μm sterile filter. The protein concentration was determined by UV spectroscopy at 280 nm and the recombinant protein was stored at 4°C.

Spiegel H., Boes A., Voepel N., Beiss V., Edgue G., Rademacher T., Sack M., Schillberg S., Reimann A, & Fischer R. (2015). Application of a Scalable Plant Transient Gene Expression Platform for Malaria Vaccine Development. Frontiers in Plant Science, 6, 1169.

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Purification of His-tagged Fusion Proteins

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The cells were suspended in 30 mL of binding buffer (20 mM sodium phosphate, 500 mM sodium chloride and 20 mM imidazole, pH 7.4) and disrupted by sonication for 30 min on ice. After centrifugation at 6000×g for 30 min at 4 °C, the supernatants were loaded onto a pre-equilibrated column of Chelating Sepharose (GE Healthcare BioScience, Sweden) charged with Ni²⁺. The column was then washed with washing buffer (20 mM sodium phosphate, 500 mM sodium chloride and 50–100 mM imidazole, pH 7.4) to remove weakly bound proteins. The fusion proteins were eluted with elution buffer (20 mM sodium phosphate, 500 mM sodium chloride and 500 mM imidazole, pH 7.4). The high-concentration imidazole was removed using a Sephadex G25 column. The peak fraction was collected and concentrated by ultrafiltration. Protein concentrations were measured by the dye-binding method of Bradford using a commercial kit (BestBio, China).

Chen H, & Jiang P. (2019). Metabolic engineering of Escherichia coli for efficient biosynthesis of fluorescent phycobiliprotein. Microbial Cell Factories, 18, 58.

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Purification and characterization of THU peptides

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Acrylamide, nickel chloride, dithiothreitol (DTT), Tris, Luria-Bertani (LB) broth, ampicillin, EDTA, imidazole, isopropyl β-D-1-thiogalactopyranoside (IPTG), phenylmethylsulfonyl fluoride (PMSF), lipopolysaccharide (LPS), MOPS, puromycin, benzamidine, sodium dodecyl sulfate, sodium azide, sodium chloride, TEMED, ammonium persulfate, glycine, and hygromycin B were obtained from Sigma-Aldrich (St. Louis, MO, USA). Chelating Sepharose and SP Sepharose were purchased from GE Healthcare (Uppsala, Sweden). Preparations of tat-derived peptide (GRKKRRQRRR)-His₆-ubiquitin (THU) and THU-RRFKEGGRGGKY (THUC) followed the methods [16 (link)].

Yu H.C., Huang K.Y., Lu M.C., Huang H.L., Liu S.Q., Lai N.S, & Huang H.B. (2017). Targeted Delivery of the HLA-B∗27-Binding Peptide into the Endoplasmic Reticulum Suppresses the IL-23/IL-17 Axis of Immune Cells in Spondylarthritis. Mediators of Inflammation, 2017, 4016802.

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