The supernatant was concentrated by ultrafiltration using an Amicon stirred cell (Merck Millipore) equipped with a 10-kDa membrane to a final volume of 40 ml and extensively dialyzed against 20 mM sodium phosphate and 150 mM NaCl, pH 7.5. The sample was added with NaCl up to 1 M final concentration and the protein was purified using a HiTrap chelating affinity column (5 ml) (GE Healthcare) previously loaded with 100 mM NiCl2 and equilibrated with 20 mM sodium phosphate, 1 M NaCl, pH 7.5. The column was washed with this buffer until the absorbance value at 280 nm was that of the buffer. rHvRNASET2 was eluted with the same buffer added with 100 mM imidazole; the fractions were equilibrated with 20 mM sodium phosphate and 150 mM NaCl, pH 7.5, by gel-permeation chromatography (PD10 column, GE Healthcare). The amount of protein was determined by using the absorbance intensity at 280 nm and a molar extinction coefficient of 66 mM−1 cm−1 (6 (link)). The recombinant rHvRNASET2 was isolated as a single band at ≈36 kDa with >90% purity as judged by SDS-PAGE analysis: ≈3.5 mg of purified enzyme per liter of fermentation broth was obtained.
Hitrap chelating affinity column
Manufactured by GE Healthcare
Sourced in United Kingdom
The HiTrap chelating affinity column is a pre-packed column designed for the purification of histidine-tagged recombinant proteins. It utilizes Ni2+ as the immobilized metal ion to selectively bind and capture the histidine-tagged proteins from complex mixtures. The column can be easily connected to a chromatography system for efficient protein purification.
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Lab products found in correlation
2 protocols using hitrap chelating affinity column
1
Purification of recombinant HvRNASET2 enzyme
2
Purification of E. coli 7α-HSDH and 7β-HSDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
E. coli cell pellets were resuspended in lysis buffer (50 mm KPi buffer, 1 m NaCl, 5 % glycerol (v/v) and 10 μg mL−1 DNAse, pH 8.0) and disrupted by French press (Constant Systems Limited, Low March, UK) (2 cycles, 180 psi). The insoluble fraction of the lysates was removed by centrifugation at 39 000 g for 30 min at 4 °C. Crude extract was loaded onto a HiTrap chelating affinity column (GE Healthcare, Little Chalfont, UK), previously loaded with Ni2+ metal ions and equilibrated with 50 mm KPi buffer, 1 m NaCl and 5 % glycerol (v/v), pH 8.0. The columns were washed with this buffer until the absorbance value at 280 nm was that of the buffer and the bound proteins were eluted with 50 mm KPi buffer, 250 mm imidazole, and 5 % glycerol (v/v), pH 8.0. The fractions containing the desired activity were dialyzed overnight against 50 mm KPi buffer and 5 % glycerol (v/v), pH 8.0, using a 3‐kDa dialysis tube. During the purification procedure, 7α‐HSDH and 7β‐HSDH activities were assayed by using the standard activity assay (see below).
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E. coli cell pellets were resuspended in lysis buffer (50 m
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