Fluorescence measurements were performed using a Tecan analyzer infinite 200Pro (Tecan Group Ltd, Männedorf, Switzerland). For mCherry, the excitation wavelength was 587 nm and the emission wavelength was 610 nm. For sfGFP and GFPmut3.1, the excitation wavelength was 485 nm and the emission wavelength was 520 nm. For quantification, calibration was performed using in‐house purified target proteins. We purified mCherry with immobilized metal affinity chromatography using His‐Tag‐specific binding on an Ni‐Sepharose HP column (GE Healthcare Bio‐Sciences, Uppsala, Sweden). GFP and sfGFP were purified through anion exchange chromatography using a Capto Q column (GE Healthcare Bio‐Sciences, Uppsala, Sweden), followed by hydrophobic interaction chromatography using a Butyl Sepharose HP column (GE Healthcare Bio‐Sciences, Uppsala, Sweden), and finally size exclusion chromatography using a Superdex 75 column (GE Healthcare Bio‐Sciences, Uppsala, Sweden). We applied the Beer–Lambert law to determine the concentrations of the standards using the absorbance at 280 nm and the corresponding excitation coefficients.
Ni sepharose hp column
Manufactured by GE Healthcare
Sourced in Sweden, United States
The Ni Sepharose HP column is a pre-packed chromatography column designed for the purification of histidine-tagged proteins. The column contains Ni Sepharose High Performance resin, which is based on cross-linked agarose beads that have been charged with nickel ions. This resin has a high binding capacity for histidine-tagged proteins, allowing for efficient capture and purification of the target proteins.
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4 protocols using ni sepharose hp column
1
Fluorescence Quantification Protocol
2
Purification of Monomeric Neon Green Protein
3
Recombinant E. coli Protein Purification
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The recombinant E. coli strains were inoculated into LB medium containing 100 μg/ml Ampicillin sodium and kept at 37°C with stirring at 200 r/min for about 3 h until the OD600 value reached 0.6–0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and then recombinant E. coli strains were kept at 16°C and 200 r/min for 20 h. The cells were collected by centrifugation at 4°C and 8,000 × g for 5 min, and were washed twice with Tris–HCl buffer (50 mM, pH 7.5). The cells were disrupted using an ultrasonic cell disrupter and were centrifuged at 4°C and 9,000 × g for 20 min to obtain the crude enzyme solution, which was used for purification and esterification in aqueous phase.
The crude enzyme was purified on a Ni Sepharose HP column (1 ml, GE, Uppsala, Sweden) using ÄKTA Fast Protein Liquid Chromatography purification system (GE, Uppsala, Sweden) with phosphate buffer (50 mM, pH 7.4) containing 400 mM NaCl and different concentrations of imidazole. The purified enzyme band was verified by SDS-PAGE, the protein concentration was determined using a BCA protein assay kit (Thermo Fischer Scientific Inc., Rockford, United States).
The crude enzyme was purified on a Ni Sepharose HP column (1 ml, GE, Uppsala, Sweden) using ÄKTA Fast Protein Liquid Chromatography purification system (GE, Uppsala, Sweden) with phosphate buffer (50 mM, pH 7.4) containing 400 mM NaCl and different concentrations of imidazole. The purified enzyme band was verified by SDS-PAGE, the protein concentration was determined using a BCA protein assay kit (Thermo Fischer Scientific Inc., Rockford, United States).
4
Recombinant ORF8 Protein Purification
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The strain (20 mL) was added in 1 L LB medium containing 100 μg/ml kalamycin and cultured for overnight with vigorous shaking at 37 °C. When the strain density was in the range of 0.6–0.8 at 600 nm, IPTG was added at a final concentration of 0.6 mM for incubation at 16℃ for 20 h. The cells were harvested and sonicated in an ice bath.
The inclusion bodies were solubilized by 50 mM Tris-HCl buffer (pH 8.0) containing 6 M guanidine hydrochloride and 10 mM reduced glutathione (buffer A). Then, the sample was loaded on a Ni Sepharose HP column (0.5 cm × 5 cm, GE Healthcare, USA). The column was eluted with 50 mL buffer A and then with 50 mL buffer A containing 0.5 M imidazole. The fractions containing ORF8 were pooled and ORF8 was in denatured state. In order to renature ORF8, the pooled solution was added dropwise to a 50-fold excess of 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 M L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione and 0.5 M urea at 4 °C. Finally, the renatured ORF8 was dialyzed against PBS buffer (pH 7.4).
The inclusion bodies were solubilized by 50 mM Tris-HCl buffer (pH 8.0) containing 6 M guanidine hydrochloride and 10 mM reduced glutathione (buffer A). Then, the sample was loaded on a Ni Sepharose HP column (0.5 cm × 5 cm, GE Healthcare, USA). The column was eluted with 50 mL buffer A and then with 50 mL buffer A containing 0.5 M imidazole. The fractions containing ORF8 were pooled and ORF8 was in denatured state. In order to renature ORF8, the pooled solution was added dropwise to a 50-fold excess of 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 M L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione and 0.5 M urea at 4 °C. Finally, the renatured ORF8 was dialyzed against PBS buffer (pH 7.4).
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