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Histrap hp ni sepharose column

Manufactured by GE Healthcare

The HisTrap HP Ni-sepharose column is a pre-packed chromatography column used for the purification of histidine-tagged proteins. It consists of Ni-Sepharose high-performance resin that selectively binds to the histidine tags present on the target proteins, allowing for their isolation from complex mixtures. The column is designed for fast and efficient protein purification, with a simple workflow that can be easily integrated into various laboratory settings.

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3 protocols using histrap hp ni sepharose column

1

Nickel-Affinity Purification of His-Tagged Proteins

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Lysates were purified using a
1 mL HisTrap HP Ni-sepharose column (GE Healthcare Life Sciences)
connected to an ÄKTA pure protein purification system (Cytiva).
The column was equilibrated with 5 CV Buffer A (50 mM Tris-HCl, 300
mM NaCl, pH 8.0) before the supernatant was loaded onto the column.
Impurities were removed by washing with Buffer A for 10 CV. His-tagged
proteins were eluted using a gradient of 0–100% Buffer B (50
mM Tris-HCl, 300 mM NaCl, 400 mM imidazole, pH 8.0) over 40 CV. All
steps were performed using a flow rate of 1 mL/min. GFP-containing
fractions were identified using 488 nm absorbance.
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2

Recombinant Hepatitis C Virus E2 Ectodomain

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The sequence encoding amino acid residues (aa) 384–661 of HCV genotype 1a strain H77c (GenBank accession no. AF009606), plus an N-terminal 6-histidine tag, was inserted into the pAcGP67-A transfer vector (BD Biosciences). Sf9 cells were co-transfected with the transfer vector and Baculogold™ linearized baculovirus DNA, to make recombinant baculovirus (rBac) expressing the ectodomain of E2 as a secreted, soluble protein. The rBac E2661 was cloned by limiting dilution, amplified, and used to infect Hi Five™ cells at a multiplicity of infection (moi) of 1. After 3 days’ incubation the supernatant medium was harvested, filtered through a 0.22 µm membrane, and dialyzed first against 20 mM sodium phosphate pH 6.8, 150 mM NaCl and subsequently against 20 mM sodium phosphate pH 8.0, 300 mM NaCl. Ultrapure imidazole was added to a concentration of 20 mM, and the supernatant loaded onto a HisTrap HP Ni Sepharose column using an Äkta Purifier (GE Healthcare). E2661 was eluted with a gradient of 40–200 mM imidazole in 20 mM sodium phosphate pH 8.0, 300 mM NaCl, to separate monomeric molecules from dimers and multimers. Monomeric E2661 was purified to homogeneity by size exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column (GE Healthcare).
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3

Affinity Purification of His-tagged Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Lysates were purified using a 1 mL HisTrap HP Ni-sepharose column (GE Healthcare Life Sciences) connected to an ÄKTA pure protein purification system (Cytiva). The column was equilibrated with 5 CV Buffer A (50 mM Tris-HCl, 300 mM NaCl, pH 8.0) before loading the supernatant onto the column.
Impurities were removed by washing with Buffer A for 10 CV. His-tagged proteins were eluted using a gradient of 0-100% Buffer B (50 mM Tris-HCl, 300 mM NaCl, 400 mM imidazole, pH 8.0) over 40 CV. All steps were performed using a flow-rate of 1 mL/min. GFP-containing fractions containing were identified using absorbance at 488 nm.
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