Histrap 5 ml

Manufactured by GE Healthcare

Sourced in United Kingdom

The HisTrap 5 mL is a pre-packed column for the rapid and efficient purification of histidine-tagged recombinant proteins. The column contains Ni Sepharose High Performance resin, which has a high binding capacity for histidine-tagged proteins. The HisTrap 5 mL column can be used with various chromatography systems and is designed for simple, fast, and reproducible purification of histidine-tagged proteins.

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Lab products found in correlation

Ti45 rotor, by Beckman Coulter (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Emulsiflex c3, by Avestin (1 mentions) Superdex 75 26 60, by GE Healthcare (1 mentions) Complete edta free, by Roche (1 mentions) Superdex 75 16 60, by GE Healthcare (1 mentions)

Close spelling variants of this product

HisTrap Excel 5-mL column HisTrap FF 5-mL affinity column HisTrap HP 5 mL HisTrap 5 ml column HisTrap HP 5 mL column HisTrap FF crude 5 ml HisTrap FF 5 ml column HisTrap

6 protocols using histrap 5 ml

Expression and Purification of Recombinant Protein

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Transformed E. coli were grown on LB plates containing kanamycin overnight at 37 °C and seeded into 500 ml Terrific Broth containing 25 μg/mL kanamycin. After incubation for 60 h at 25 °C, the samples were centrifuged at 4,000 rpm for 15 min and 4 °C. The resulting pellet was resuspended in binding buffer (500 mM NaCl, 30 mM imidazole) and thrice passed through a homogenizer (EmulsiFlex-C3, AVESTIN Inc.; Ottawa, Canada) at 1,500 bar. The cell debris was removed from the supernatant by centrifuging twice at 16,000 g for 20 min at 15 °C, whereas the supernatant was applied to a nickel affinity column (HisTrap 5 ml; GE Healthcare; Buckinghamshire, United Kingdom) primed with the binding buffer.
Elution was performed using a linear gradient with an appropriate buffer (500 mM NaCl, 500 mM imidazole). The fractions containing TG were collected and dialyzed against a phosphate buffer at 4 °C (50 mM sodium phosphate buffer pH 8). Protein concentration was calculated with a NanoDrop 2000 (Thermo Fisher Scientific Inc. Massachusetts, USA) using a molar extinction coefficient of 71,850 1/M⋅cm and molecular weight of 39.07 kDa, calculated using the ProtParam (Swiss Institute of Bioinformatics) online software.

Javitt G., Ben-Barak-Zelas Z., Jerabek-Willemsen M, & Fishman A. (2017). Constitutive expression of active microbial transglutaminase in Escherichia coli and comparative characterization to a known variant. BMC Biotechnology, 17, 23.

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Purification of E. coli DnaJ and DnaK

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E.coli pellets were suspended in a 50-mM phosphate buffer (pH 6.5 for DnaJs and pH 7.5 for DnaK), with 150-mM NaCl, 1-mM PMSF, and 3-mM β-ME. Cells were disrupted by sonication and centrifuged (30,000 g, 30 min, 4 °C). The clarified lysate was then loaded on a Ni‐NTA resin (HisTrap 5 mL, GE Healthcare), equilibrated with a binding buffer (50-mM phosphate buffer, pH 6.5 for DnaJs, pH 7.5 for DnaK, 150-mM NaCl, 10-mM imidazole), and bound proteins were eluted using a gradient of imidazole (10–300 mM). Fractions containing DnaJ were pooled and dialyzed against the same buffer but without imidazole. Finally, the buffer was supplemented with 5% glycerol, and the protein was stored at -80 ºC. For DnaK, after the HisTrap step, fractions containing DnaK were pooled and loaded on a Superdex column 200 10/300, equilibrated with 50-mM phosphate buffer, pH 7.5, with 150-mM NaCl. Isocratic elution of proteins was done using the Akta Purifier system. Fractions containing pure DnaK2 were pooled, supplemented with 5% glycerol, and stored at -80 ºC.

Rydzy M., Kolesiński P., Szczepaniak A, & Grzyb J. (2023). DnaK and DnaJ proteins from Hsp70/40 family are involved in Rubisco biosynthesis in Synechocystis sp. PCC6803 and sustain the enzyme assembly in a heterologous system. BMC Plant Biology, 23, 109.

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Co-production of PXR-LBD and SRC-1 for Stability

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The human PXR-LBD (130-434) was co-produced with a fragment of the steroid receptor coactivator-1 (SRC-1, 623-710) to enhance PXR stability. The PXR-LBD gene was cloned into pRSET-A with a His₆ tag at the N-terminus (gift from Matthew Redinbo, University of North Carolina at Chapel Hill, USA), and the SRC-1 fragment gene has been inserted into the pACYC184 vector. Proteins were overproduced in E. coli BL21(DE3) cells overnight at 20 °C in LB medium without any ligand. After culture cells were harvested by centrifugation and the pellets resuspended in lysis buffer (20 mM Tris pH 7.5, 250 mM NaCl, 5% (v/v) glycerol) supplemented with lysozyme (1 μg ml⁻¹) and a protease inhibitor cocktail tablet (cOmplete, EDTA-free, Roche), and then subjected to sonication. The clarified cell lysate was applied onto a Ni²⁺-affinity column (HisTrap 5 ml; GE Healthcare) equilibrated with lysis buffer supplemented with 10 mM imidazole. The eluted PXR-LBD was then applied onto a gel filtration column (Superdex 75 26/60; GE Healthcare) equilibrated with a buffer containing 20 mM Tris pH 7.8, 250 mM NaCl, 5% (v/v) glycerol, 5 mM DTT, 1 mM EDTA. The PXR-LBD was concentrated and stored at −40 °C.

Delfosse V., Dendele B., Huet T., Grimaldi M., Boulahtouf A., Gerbal-Chaloin S., Beucher B., Roecklin D., Muller C., Rahmani R., Cavaillès V., Daujat-Chavanieu M., Vivat V., Pascussi J.M., Balaguer P, & Bourguet W. (2015). Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds. Nature Communications, 6, 8089.

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High-Yield Production of HPBP Proteins

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Productions of HPBP and sHPBP were performed using E. coli BL21(DE3)-pGro7/GroEL cells (TaKaRa) in 6 l of ZYP medium [1 (link)] (100 μg ml⁻¹ ampicillin, 34 μg ml⁻¹ chloramphenicol). The cultures were grown at 37 °C to reach OD_600nm = 0.6 and then induced by starting the consumption of lactose in ZYP medium coupled to temperature transition to 17 °C during 16 h. Cells were harvested by centrifugation (4500g, 4 °C, 15 min) and pellets were suspended in 400 ml of lysis buffer (20 mM TRIS, pH 8, 100 mM NaCl, Lysozyme 0.25 mg ml⁻¹, DNAse I 10 μg ml⁻¹, PMSF 0.1 mM, MgSO₄ 20 mM and 8 tablets of anti-protease EDTA-free (Roche)) and stored at −80 °C for 2 h. Cells were then thawed at 37 °C for 15 min and disrupted by 3 steps of 30 s of sonication (QSonica sonicator; amplitude 40). Debris was removed by centrifugation (12,500g, 4°C, 30 min). Supernatant was loaded on a Nickel affinity column (HisTrap 5 ml, FFCrude from GE Healthcare) at a flow rate of 5 ml min⁻¹. Proteins gripped to the column were eluted by imidazol, using an elution buffer (20 mM TRIS, pH 8, 100 mM NaCl and 250 mM imidazole). Then, a size exclusion chromatography step (Superdex 75 16/60, GE Healthcare) was performed using buffer 20 mM TRIS, pH 8 and 100 mM NaCl. Protein production and purity were checked by 15% SDS–PAGE analysis (Fig. 1-5) and mass spectrometry analysis (Plateforme Timone, Marseille, France).

Gonzalez D., Hiblot J., Darbinian N., Miller J.C., Gotthard G., Amini S., Chabriere E, & Elias M. (2014). Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein. FEBS Open Bio, 4, 121-127.

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Purification of Tagged Rubisco from E. coli

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Tagged Rubisco was purified from E. coli BL21 DE3(Star) cells transformed with plasmids pET11a‐AtC60αβ/C20, pDUET‐AtR1/R2/RX/B2, and pET28‐AtRbcLS^His6 and expressed in 6 L LB media, as described above, in the presence of 0.3 mm IPTG for 10 h at 23 °C. The cell pellet was resuspended in extraction buffer (30 mm Tris pH 7.9, 150 mm NaCl, 10 mm imidazole, 0.5 g·L⁻¹ lysozyme, 5 U·mL⁻¹ benzonase, 5% glycerol), disrupted by sonication and centrifuged (70 000 g, 30 min, 4 °C, TI45 rotor; Beckman Coulter). The clarified lysate was then loaded on a nickel‐nitrilotriacetic acid (Ni‐NTA) column (HisTrap 5 mL; GE Healthcare) equilibrated with NTA buffer (30 mm Tris pH 7.9, 150 mm NaCl, 10 mm imidazole, 5% glycerol), and bound proteins were eluted using a gradient of imidazole (10–300 mm). Fractions containing Rubisco were pooled and loaded on a HiPrep 26/60 Sephacryl S‐300 HR column equilibrated with SEC buffer. Fractions containing Rubisco were then pooled, concentrated, and stored as above.

Wilson R.H., Thieulin‐Pardo G., Hartl F, & Hayer‐Hartl M. (2019). Improved recombinant expression and purification of functional plant Rubisco. Febs Letters, 593(6), 611-621.

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Purification and Characterization of AtRcaβ

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The β isoform of Rubisco activase from A. thaliana (AtRcaβ) was purified from E. coli BL21 DE3(Star) cells transformed with plasmid pET28‐Ub‐AtRcaβ and plasmid pEcEL/ES and expressed in LB medium. E. coli cells were resuspended in extraction buffer (30 mm Tris pH 7.9, 150 mm NaCl, 10 mm imidazole, 0.5 g·L⁻¹ lysozyme, 5 U·mL⁻¹ Benzonase, 5% glycerol), disrupted by sonication and centrifuged (70 000 g, 30 min, 4 °C, TI45 rotor; Beckman Coulter). The clarified lysate was then loaded on a Ni‐NTA column (HisTrap 5 mL; GE Healthcare) equilibrated with NTA buffer, and bound proteins were eluted using an imidazole gradient of 10–300 mm. Fractions containing Rubisco activase were pooled, treated by the deubiquitinase enzyme Usp2 (0.5 mg) 23 in presence of 0.05% β‐mercaptoethanol for 3 h to remove the ubiquitin tag and loaded on a HiPrep 26/60 Sephacryl S‐300 HR column equilibrated with SEC buffer. Fractions containing AtRcaβ were pooled, concentrated, and stored as above.
All purified protein were quantified by absorbance at 280 nm using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) using extinction coefficients of 103 250 m⁻¹·cm⁻¹ for AtRbcLS (MW 73 153.2 Da) and AtRbcLS^His6 (MW 74 289.4 Da), and 36 270 m⁻¹·cm⁻¹ for AtRcaβ (43 308 Da).

Wilson R.H., Thieulin‐Pardo G., Hartl F, & Hayer‐Hartl M. (2019). Improved recombinant expression and purification of functional plant Rubisco. Febs Letters, 593(6), 611-621.

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