The recombinant VEGF-A109, VEGF-A165, VamminKPRR and VamminPRRK proteins were produced in adherent HEK293T cells by calcium phosphate transfection with plasmids encoding N-terminally His-tagged proteins. On the following day, DMEM (Sigma-Aldrich) supplemented with 10% FBS was replaced with serum free media and cells were incubated for 2 days. The proteins were purified using HisTrapExcel columns with ÄktaAvant (GE Healthcare) using gradient elution from 0 to 500 mM Imidazole in PBS. The collected fractions were analyzed by SDS-PAGE. Pure fractions were pooled, diluted to PBS and concentrated using Amicon Ultra-15 3 K Centrifugal Filter Units and final buffer exchange to PBS was performed using HiTrap Desalting columns (GE Healthcare). Protein concentration was assessed using a BCA Protein Assay Kit (Thermo Scientific) and verified by SDS-PAGE. Recombinant Vammin, sVEGFR2-Fc and sNRP1-Fc were produced as described earlier17 (link).
Histrap excel column
Manufactured by GE Healthcare
Sourced in United States, Sweden, Germany
The HisTrap Excel column is a pre-packed affinity chromatography column designed for the purification of histidine-tagged proteins. It utilizes a highly selective and robust ligand for the capture and purification of these target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Expi293f cells, by Thermo Fisher Scientific (5 mentions)
Expi293 expression system, by Thermo Fisher Scientific (5 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (4 mentions)
Sephadex g 25 medium, by GE Healthcare (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (3 mentions)
Streptrap hp column, by GE Healthcare (2 mentions)
Freestyle293 cells, by IBA Lifesciences (2 mentions)
Streptrap hp column, by IBA Lifesciences (2 mentions)
293 freestyle cells, by Thermo Fisher Scientific (2 mentions)
Biolock biotin blocking solution, by IBA Lifesciences (2 mentions)
Expi293 cell, by Thermo Fisher Scientific (2 mentions)
Tskgel g4000swxl, by Tosoh (2 mentions)
Endoglycosidase f1, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (2 mentions)
Superdex 200 10 300 column, by GE Healthcare (2 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (2 mentions)
Hiprep 26 10 column, by GE Healthcare (2 mentions)
75 protocols using histrap excel column
1
Production and Purification of Angiogenic Proteins
2
Purification of Glyco_hydro_20 Hexosaminidase
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Example 3
The His-tagged Glyco_hydro_20 hexosaminidase enzyme was purified by immobilized metal chromatography (IMAC) using Ni2+ as the metal ion on 5 mL HisTrap Excel columns (GE Healthcare Life Sciences). The purification took place at pH 7 and the bound protein was eluted with imidazole. The purity of the purified enzyme was checked by SDS-PAGE and the concentration of the enzyme determined by Absorbance 280 nm after a buffer exchange in 50 mM HEPES, 100 mM NaCl pH 7.0.
3
His-tag Purification of Recombinant Leishmania Proteins
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His6-tag fused, recombinant proteins secreted by transfected Leishmania cells were purified by liquid chromatography over His-Trap™ excel columns (GE Healthcare, Buckinghamshire, United Kingdom). Protein-containing fractions were pooled and dialyzed two times against PBS (4 h and overnight). Finally, protein concentrations were assessed using Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Rockford, USA). Identity of purified, recombinant proteins was verified by LC-MSMS analysis after tryptic digestion.
4
Purification of His-tagged Enzymes
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Example 2
All His-tagged enzymes were purified by immobilized metal chromatography (IMAC) using Ni2+ as the metal ion on 5 mL HisTrap Excel columns (GE Healthcare Life Sciences). The purification took place at pH 8 and the bound proteins were eluted with 50 mM HEPES, pH 7.0 and 0.75 M imidazole. Subsequently, the enzyme sample was desalted by loading onto a Sephadex™ G-25 (medium) (GE Healthcare, Piscataway, N.J., USA) column equilibrated in 50 mM HEPES pH 7.0, 100 mM NaCl and eluting with the same buffer. The purity of the purified enzymes was checked by SDS-PAGE and the concentration of each enzyme determined by Abs 280 nm after a buffer exchange.
5
Purification of His-tagged Hexosaminidase Enzyme
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Example 3
The His-tagged Glyco_hydro_20 hexosaminidase enzyme was purified by immobilized metal chromatography (IMAC) using Ni2+ as the metal ion on 5 mL HisTrap Excel columns (GE Healthcare Life Sciences). The purification took place at pH 7 and the bound protein was eluted with imidazole. The purity of the purified enzyme was checked by SDS-PAGE and the concentration of the enzyme determined by Absorbance 280 nm after a buffer exchange in 50 mM HEPES, 100 mM NaCl pH7.0
6
SARS-CoV-2 Spike Protein Production
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We previously used a prefusion-stabilized, trimeric spike ectodomain (S2Pecto) to structurally define the sites several antibodies recognized on the SARS-CoV-2 spike trimer (Zost et al., 2020a (link)). This construct is similar to ones previously reported (Wrapp et al., 2020 (link)) and includes the ectodomain of SARS-CoV-2 (to residue 1,208), a T4 fibritin trimerization domain, and C-terminal 8x-His tag and TwinStrep tags. The construct also includes K986P and V987P substitutions to stabilize the spike in the prefusion conformation and a mutated furin cleavage site. S2Pecto protein was expressed in FreeStyle 293 cells (ThermoFisher) or Expi293 cells (ThermoFisher). Expressed S2Pecto protein was isolated by metal affinity chromatography on HisTrap Excel columns (GE Healthcare), followed by further purification on a StrepTrap HP column (GE Healthcare) and size-exclusion chromatography on TSKgel G4000SWXL (TOSOH). We also expressed a recently reported spike protein construct with 4 additional proline substitutions that enhance thermostability, yield, and structural homogeneity, here referred to as S6Pecto (Hsieh et al., 2020 (link)). The S6Pecto protein was expressed in FreeStyle293 cells and isolated on a StrepTrap HP column following the addition of BioLock Biotin Blocking Solution (IBA Lifesciences) to the culture supernatant.
7
Structural Characterization of SARS-CoV-2 Spike
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We previously used a prefusion-stabilized, trimeric spike ectodomain (S2P ecto) to structurally define the sites several antibodies recognized on the SARS-CoV-2 spike trimer (Zost et al., 2020a (link)). This construct is similar to ones previously reported (Wrapp et al., 2020 (link)) and includes the ectodomain of SARS-CoV-2 (to residue 1,208), a T4 fibritin trimerization domain, and C-terminal 8x-His tag and TwinStrep tags. The construct also includes K986P and V987P substitutions to stabilize the spike in the prefusion conformation and a mutated furin cleavage site. S2P ecto protein was expressed in FreeStyle 293 cells (ThermoFisher) or Expi293 cells (ThermoFisher). Expressed S2P ecto protein was isolated by metal affinity chromatography on HisTrap Excel columns (GE Healthcare), followed by further purification on a StrepTrap HP column (GE Healthcare) and size-exclusion chromatography on TSKgel G4000SW XL (TOSOH). We also expressed a recently reported spike protein construct with 4 additional proline substitutions that enhance thermostability, yield, and structural homogeneity, here referred to as S6P ecto (Hsieh et al., 2020 ). The S6Pecto protein was expressed in FreeStyle293 cells and isolated on a StrepTrap HP column following the addition of BioLock Biotin Blocking Solution (IBA Lifesciences) to the culture supernatant.
8
SARS-CoV-2 Spike and RBD Protein Purification
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Culture supernatants were applied using a peristaltic pump to HisTrap excel columns (GE Healthcare) with a flow rate of 2–3 ml/min, using 1 ml of media for each 100 ml of supernatant. Columns were washed with 10 volumes of buffer A (NaH2PO4 57,5mM, NaCl 300 mM, pH 7.8) and next with 60 volumes of buffer A with 10 mM imidazol. Then, columns were connected to an HPLC system (Kanuer) and washed at 2 ml/min with the last buffer until absorbance at 280 nm reached a value below 0.005. Proteins were eluted with a 12 minute linear gradient from 10 to 1 M imidazole in buffer A. Fractions of 1 ml were collected and analyzed by SDS-PAGE. Buffer of fractions containing protein was exchange to PBS with NAP-5 columns (GE Healthcare) and pooled. Typical yields were around 6 and 45 mg/l for spike and RBD, respectively, with a purity level of at least 98%.
9
Purification and Characterization of Dsg2 Extracellular Domains
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The extracellular regions EC1-EC4 of Dsg2 were stably transfected in HT1080 cells as previously reported26 (link). Expressed Dsg2 was purified from the cell culture supernatant by affinity chromatography on HisTrap excel columns (GE Healthcare, Solingen, Germany), according to the manufacturer’s instructions. The protein solutions were concentrated with Amicon Ultra-15 Centrifugal Filter Units (10,000 MWCO, MerckMillipore, Darmstadt, Germany). Proteins were identified by Western blotting and analyzed by means of SDS-Page with Coomassie-brilliant-blue staining, revealing a purity of at least 90%. The concentration of Dsg2 varied between 0.8 gL−1 1.5 gL−1.
10
His-tagged Enzyme Purification by IMAC
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Example 2
All His-tagged enzymes were purified by immobilized metal chromatography (IMAC) using Ni2+ as the metal ion on 5 mL HisTrap Excel columns (GE Healthcare Life Sciences). The purification took place at pH 8 and the bound proteins were eluted with 50 mM HEPES, pH7.0 and 0.75 M imidazole. Subsequently, the enzyme sample was desalted by loading onto a Sephadex™ G-25 (medium) (GE Healthcare, Piscataway, N.J., USA) column equilibrated in 50 mM HEPES pH 7.0, 100 mM NaCl and eluting with the same buffer. The purity of the purified enzymes was checked by SDS-PAGE and the concentration of each enzyme determined by Abs 280 nm after a buffer exchange.
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