Fetuin-A (Fet-A) was purified from commercially available bovine fetuin (FETB-16-N-1, Alpha Diagnostic International, San Antonio, TX, USA). First, size exclusion chromatography was performed with ÄKTApurifier (GE Healthcare, Chicago, IL, USA) using a HiPrep 26/60 Sephacryl S-200 HR column and phosphate buffer saline. Then, Fet-A was further purified by ionexchange chromatography with a 5 mL HiTrap Q HP. Buffer A (20 mM N-methylpiperazine, pH 4.8) and buffer B (20 mM N-methylpiperazine, 1 M NaCl, pH 4.8) were used as running buffers. Proteins bound to the column were eluted by a linear gradient of 0–30% buffer B. Fractions from the size exclusion chromatography and the ionexchange chromatography were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining.
Hiprep 26 60 sephacryl s 200 hr column
Manufactured by GE Healthcare
Sourced in United States
The HiPrep 26/60 Sephacryl S-200 HR column is a size exclusion chromatography column designed for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. The column is packed with a high-resolution Sephacryl S-200 HR matrix, which provides efficient separation within a wide molecular weight range.
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Lab products found in correlation
Akta purifier, by GE Healthcare (2 mentions)
Kta purifier, by GE Healthcare (1 mentions)
Ni nta column, by Thermo Fisher Scientific (1 mentions)
Centrifugal filtration, by Merck Group (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
Amicon ultra 15 centrifuge filter unit, by Merck Group (1 mentions)
Cellulose membrane of 14 kda, by Merck Group (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
6 protocols using hiprep 26 60 sephacryl s 200 hr column
1
Bovine Fetuin-A Purification Protocol
2
Recombinant Expression and Purification of PDE5A
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The catalytic domain of PDE5A (residues 535-862; GenBank accession number BC126233.1) was subcloned into the T7 promoter-driven expression vector pET21b with a 6 × His-tag at the C-terminus (Hsieh et al., 2020 (link)). The recombinant plasmid was transformed into E. coli strain BL21 (DE3) and grown in an autoinducing medium (Studier 2005 (link)) containing 50 μg/mL ampicillin at 37 C until OD600 = 0.6–0.7, then induced protein expression at 15°C for 40 h. The PDE5A protein was purified through the Ni-NTA column (Thermo Scientific) and further purified by the HiPrep™26/60 Sephacryl™-S-200HR column (GE Healthcare). A typical batch cell yielded over 10 mg of the PDE5A protein from 1 L of autoinducing medium with a purity >95% based on SDS‒PAGE. The protein was concentrated to a certain concentration using centrifugal filters and stored in a storage buffer (50 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM β-mercaptoethanol, 1 mM EDTA, and 5% glycerol).
3
Recombinant TRL-1 Protein Purification
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trl-1 was cloned into the pETduet vector to produce His6-tag–fused recombinant protein. The fused protein was expressed in E. coli Rosetta induced by 1 mM IPTG for 16 h at 16 °C. To purify His-tagged TRL-1, we resuspended the E. coli sediments in binding buffer (20 mM Hepes pH 7.5, 500 mM NaCl, and 10 mM imidazole), lysed with a high-pressure homogenizer, and sedimented at 18,000 rpm, 30 min at 4 °C. The supernatants were incubated with Ni-NTA agarose beads (QIAGEN) and washed with extensive binding buffers. The proteins were then eluted with His6 elution buffer (20 mM Hepes pH 7.5, 500 mM NaCl, and 500 mM imidazole) and further purified with a HiPrep 26/60 Sephacryl S-200 HR column (GE Healthcare) on an AKTA purifier (GE Healthcare). The proteins were finally eluted with a buffer containing 20 mM Hepes pH 7.5, 500 mM NaCl, concentrated by centrifugal filtrations (Millipore), and then stored in aliquots at −80 °C.
4
Purification of MBP-tagged Recombinant Proteins
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All genes were PCR-amplified and cloned into the pETMBP.3C vector to produce MBP-tag-fused recombinant proteins. Deletions were introduced by the site-directed mutagenesis approach. All recombinant proteins used in this study were expressed in E. coli BL21-CodonPlus (DE3) with induction by 1 M IPTG for 16 h at 18°C. Following induction, E. coli cells were resuspended in binding buffer (50 mM Tris-Cl, pH 7.9, 1 M NaCl, and 10 mM imidazole), lysed with a high-pressure homogenizer, and sedimented at 18,000 rpm for 30 min. The supernatant lysates were purified on Amylose Resin (NEB). After extensive washing with binding buffer, the proteins were eluted with MBP elution buffer (50 mM Tris-Cl, pH 7.9, 1 M NaCl, and 10 mM imidazole for MBP-tagged proteins), then purified with a HiPrep 26/60 Sephacryl S-200 HR column (17-1195-01; GE Healthcare) on an AKTA purifier (GE Healthcare), and eluted with a buffer containing 20 mM HEPES, pH 7.4, 500 mM NaCl.
5
Purification of Fusion Proteins by SEC
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The fusion proteins were purified by size exclusion chromatography (SEC) using an AKTA Purifier UPC 100 system (GE Healthcare, Wauwatosa, WI, USA) with a Hiprep 26/60 Sephacryl S-200 HR column (60 cm × 26 mm) (GE Healthcare, Wauwatosa, WI, USA). The proteins were eluted with 30 mM Tris, 100 mM NaCl, pH 6.8. The flow rate was 1.0 mL/min and the detection wavelength was set at 280 nm. The protein eluted in the first peak was collected and concentrated on Amicon Ultra-15 centrifuge filter units with a molecular mass cutoff of 50 kDa (Millipore, Billerica, MA, USA). After a final buffer exchange to a buffer containing 10 mM histidine, 260 mM glycine, 1% sucrose, 0.005% Tween 80, the purified fusion protein was stored at −80 °C until use.
6
Protease Purification and Characterization
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Protease purification was performed as previously defined and standardized by Alves et al. (2016) . A cell suspension containing the bacteria in the exponential phase was inoculated (1%) in LB broth, incubated for 18 h, and centrifuged at 10,000 × g for 15 min at 4°C. The enzyme was isolated from crude supernatant and partially purified by fractional precipitation using ammonium sulfate (40-60%) for 1 h followed by centrifugation at 12,000 × g for 30 min at 4°C. The pellet was resuspended in 10 mM Tris-HCl, 50 mM NaCl buffer (pH 7.5) and dialyzed twice against this buffer using a cellulose membrane of 14 kDa (Sigma-Aldrich) for 8 h. All steps were performed at 4°C, and an aliquot was collected to monitor enzyme activity.
The dialyzed solution containing the enzyme was applied in a HiPrep 26/60 Sephacryl S-200 HR column (GE Healthcare, Buckinghamshire, UK) equilibrated with 10 mM Tris-HCl, 50 mM NaCl buffer (pH 7.5) using an AKTA Purifier chromatographer (GE Healthcare). Samples were collected at 1.6 mL/min and monitored by absorbance at 280 nm. Fractions containing proteolytic activity were concentrated by ultrafiltration in Amicon Ultra-15 (Millipore, Cork, Ireland) and stored at 4°C. Purification steps were monitored by SDS-PAGE, and protease activity was monitored by zymogram.
The dialyzed solution containing the enzyme was applied in a HiPrep 26/60 Sephacryl S-200 HR column (GE Healthcare, Buckinghamshire, UK) equilibrated with 10 mM Tris-HCl, 50 mM NaCl buffer (pH 7.5) using an AKTA Purifier chromatographer (GE Healthcare). Samples were collected at 1.6 mL/min and monitored by absorbance at 280 nm. Fractions containing proteolytic activity were concentrated by ultrafiltration in Amicon Ultra-15 (Millipore, Cork, Ireland) and stored at 4°C. Purification steps were monitored by SDS-PAGE, and protease activity was monitored by zymogram.
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