Fucoidan from F. evanescens (500 mg) was enzymatically hydrolyzed in optimal working conditions using 100 mg·L−1 Fhf1, and 10 mM CaCl2 in Tris-HCl buffer, at pH 8 and 37 °C. After 72 h, the reaction was stopped by heating at 80 °C for 10 min. The high molecular weight reaction products (HMP) were precipitated with ice-cold ethanol at a ratio of 1:3 (v/v) and separated by centrifugation at 10,000× g for 15 min. The supernatant containing low molecular weight reaction products (LMP) was concentrated under vacuum and applied to a Q-Sepharose HP column (1 × 10 cm) (GE Healthcare, Chicago, IL, USA) equilibrated with water. Oligosaccharides were eluted with sequential linear gradients from 0–0.75 M, from 0.5–1.5 M, and from 1–2 M NH4HCO3, at a flow rate of 1 mL·min−1. The fractions containing the carbohydrates were pooled, concentrated, and desalted by vacuum evaporation or Sephadex G-10 column. The carbohydrates in the fractions were detected using the phenol-sulphuric acid method [46 (link)]. Fractions containing carbohydrates were analyzed by C-PAGE electrophoresis, as described previously [47 (link)]. Another hydrolysis reaction using a fresh enzyme was performed on precipitated and re-solubilized HMP to ensure there was no further hydrolysis.
Q sepharose hp column
Manufactured by GE Healthcare
Sourced in United States
The Q Sepharose HP column is a chromatography column used for the purification of biomolecules. It is designed for high-performance ion exchange chromatography. The column is packed with Q Sepharose, a strong anion exchange medium.
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9 protocols using q sepharose hp column
1
Fucoidan Enzymatic Hydrolysis and Oligosaccharide Purification
2
Purification of Recombinant SNM1A Variants
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SNM1ANΔ698, SNM1A NΔ698(D736A/H737A) and SNM1ANΔ608 were expressed in Star pRARE pLysS E. coli (Invitrogen), and induced at 0.700 OD600 with 1 mM IPTG at 25°C overnight. Cells were resuspended in Nickel A buffer (20 mM HEPES pH 7.5, 1 M NaCl, 30 mM imidazole, 0.5 mM TCEP, 10% glycerol) with protease inhibitors and lysed with four passes through a French press at 10 000 psi. The lysate was clarified by centrifugation and filtration. The sample was loaded onto a HisTrap Fast Flow nickel-chelating column (GE Healthcare) and eluted with 300 mM imidazole. The sample was diluted to 250 mM NaCl using Ion Exchange Buffer A (20 mM HEPES pH 7.5, 0.5 mM TCEP, 10% glycerol) then loaded onto a Q-sepharose HP column (GE Healthcare). SNM1A was eluted with a linear gradient from 250 to 650 mM NaCl. After pooling SNM1A-containing fractions, the sample was diluted to 250 mM NaCl with Ion Exchange Buffer A and treated with TEV protease overnight to remove the His6-NusA fusion protein. The sample was then run through an SP-HP sepharose column (GE Healthcare) and eluted with a linear gradient from 250 to 750 mM NaCl. Purified Pso2 was prepared as previously described in (25 (link)).
3
Purification of T Cell Inhibitory Factors
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Anion Exchange chromatography: Spn lysate was filtered and NaCl (Sigma) added to a final concentration of 10 mM. Lysate was then loaded onto a Q Sepharose HP column (GE Lifesciences). Proteins were eluted with an increasing gradient of NaCl (10 mM to 1 M) and fractions collected. Hydroxyapatite chromatography: Fractions from the previous step that contained inhibitory activity in our functional assay were pooled. Potassium phosphate (KPi, Sigma) was added to pooled fractions to a final concentration of 10 mM. These fractions were loaded onto a hydroxyapatite column (Macro-Prep ceramic hydroxyapatite resin, type 1, 40 µM, Bio-Rad). Proteins were eluted with a 10–500 mM KPi gradient; fractions were collected and tested for inhibition of T cell effector function.
4
Recombinant DERA Expression in E. coli
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Recombinant expression of DERA was performed in 3 l baffled Fernbach flasks using E. coli BL21(DE3) cells. Chemically competent cells were transformed with the respective DERA-vectors, followed by selection on LB plates supplemented with ampicillin. 1 l terrific broth (TB) with 100 μg ml-1 ampicillin was inoculated with a 5 ml overnight culture prepared from a single colony and grown at 37°C. After incubation at 25°C and 120 rpm for 8 h, expression of deoC gene was induced by the addition of 0.1 mM IPTG. After prolonged incubation of 15 h at 25°C, cells were harvested. For disruption using Ultrasonic Desintegrator Sonoplus HP 2070 (Bandelin, Berlin), the cells were suspended 20% (w/v) in triethanol amine buffer (100 mM, pH 7). Centrifugation at 18,000 rcf and 4°C resulted in cell free crude extract. For purification 2 ml of the cell free crude extract were loaded on a Q Sepharose HP column (5 ml) applied in an ÄKTA Purifier System (GE Healthcare, Munich) and eluted with triethanol amine buffer (100 mM, pH 7). The eluate was concentrated at 4°C using Vivaspin 20 MWCO 10 kDa ultra-concentrators (Sartorius Stedim Biotech S.A., France).
5
Purification of Protein Fraction III
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Fraction III was suspended in 6 ml buffer I, dialyzed against 4 l of buffer I for 3 h, and then diluted to a conductivity of <30 mM NaCl (ca. 25 ml) prior to loading a 4 ml Q-sepharose HP column (GElifesciences.com, 0.6 × 15 cm) pre-equilibrated with buffer I + 20 mM NaCl. The column was run at a flow rate of 0.25 ml/min. After loading, the column was washed with 10 column volumes of buffer I + 20 mM NaCl. Proteins were eluted with a 10 column volume gradient of buffer I + 20 mM NaCl to buffer I + 350 mM NaCl. Fractions that contained at least 50% of the activity of the peak fraction were pooled and precipitated with ammonium sulfate (0.436 g ammonium sulfate added to each ml of solution, 22 000 × g, 45 min) and stored at 4°C as Fraction IV.
6
Succinylation of Streptavidin
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Example 2
Streptavidin is succinylated as shown in
7
Purification and Analysis of Hsp90 Variants
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Hsp90 variants with N-terminal His6 tags were bacterially expressed, purified and analyzed as described previously 23 (link). In brief, protein purification was performed using Co+NTA agarose (Qiagen), followed by a Phenyl Sepharose column, and a Q sepharose HP column (GE). Aha1 and Hch1 were purified as previously described42 (link),44 (link). The concentrations of these highly purified proteins were determined spectroscopically using extinction coefficients based on amino acid composition.
8
Succinylation of Streptavidin for Purification
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Example 2
Streptavidin is succinylated as shown in
9
Purification of Strep- or His-tagged MoFe Proteins
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All purification of Strep- or His-tagged MoFe proteins was carried out as described in detail previously (21 (link)), using either Strep-Tactin (IBA Lifesciences, Göttingen, Germany) or immobilized metal affinity (GE Healthcare) columns. When anion-exchange chromatography was used for further purification, ∼20 mg of MoFe protein isolated using either the STAC or IMAC procedure was applied to and eluted from a 1-ml Q-Sepharose HP column (GE Healthcare), using a 200–500 mm NaCl gradient, 20-ml total volume.
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