Recombinant sialylated AIM-A1 fragment, consisting of VWF residues 1238–1493 and C-terminal 10x histidine, referred to herein as sAIM-A1, was produced in BHK cells as described [22 (link)]. Media was collected and loaded onto a HisTrap Excel 5-mL column (GE Healthcare, Chicago, IL). sAIM-A1 was eluted using the buffer containing 150mM NaCl, 20mM phosphate, and 500mM imidazole at pH 7.4. The protein was further purified on a Superdex 200 16/600 preparatory grade (pg) gel filtration column in 1X phosphate-buffered saline (PBS). Positive fractions were pooled and concentrated using a Vivaspin20 concentrator unit (Sartorius, Göttengin, Germany) with a molecular weight cut-off of 10 kDa. The protein was analyzed on a 4–20% SDS-PAGE gel stained with GelCode Blue Stain for verification of purity.
Histrap excel 5 ml column
Manufactured by GE Healthcare
Sourced in United States
The HisTrap Excel 5-mL column is a pre-packed affinity chromatography column designed for the purification of histidine-tagged proteins. The column contains Ni Sepharose High Performance resin, which has a high binding capacity for histidine-tagged proteins. The column can be used with common laboratory buffers and is compatible with a variety of protein expression systems.
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Lab products found in correlation
Bradford protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Kta protein purification system, by GE Healthcare (1 mentions)
Sephadex g 25 column, by GE Healthcare (1 mentions)
Hitrap protein a 5 ml column, by GE Healthcare (1 mentions)
Hitrap igm purification hp, by GE Healthcare (1 mentions)
Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
6 protocols using histrap excel 5 ml column
1
Recombinant Sialylated AIM-A1 Purification
2
Production and Purification of FucA1 Mutants
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These mutants were produced on a smaller scale and the protocol deviates slightly from wild-type protein owing to this. Conditioned media (600 mL) was clarified and purified by His-tag affinity identically to the wild-type protein. Protein fractions containing FucA1 were pooled and dialysed against wild-type SEC buffer with in-house TEV protease overnight at ambient temperature. Dialysed samples were passed through a HisTrap excel 5-mL column (GE Healthcare) equilibrated in buffer A to remove the N-terminal His-tag and His-tagged TEV protease. Flowthroughs were collected and concentrated using 30-kDa Vivaspin concentrators before samples were further purified by SEC using a Superdex 200 Increase 10/300 GL column in SEC buffer. FucA1-containing fractions were concentrated as before and flash frozen in liquid nitrogen. Samples were stored at −80°C before use. Purity of samples was assessed by SDS-PAGE (Extended Data Figures S8 and S12 ).
3
Recombinant Protein Purification from E. coli
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E. coli BL21(DE3) harboring pETDuet-Bga1903+ was grown aerobically in LB, supplemented with 100 μg/mL ampicillin, at 37 °C to an OD600 ≈ 0.8. At the time, IPTG was added into the culture to a final concentration of 1 mM and the cultivation was continued at 28 °C for 18 h. The culture was centrifuged at 10,000× g for 15 min at 4 °C, and the cell-free supernatant was harvested. This protein solution was 100-fold concentrated by using the Millipore Labscale TFF system, equipped with Pellicon XL cassette (Biomax 5 kDa). The concentrated solution was loaded into a HisTrap excel 5 mL column (GE Healthcare Life Science, Marlborough, MA, USA), followed by an intensive wash with PBS buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 7.4). Finally, the protein bound on the resin was eluted with 100 mM imidazole-containing PBS buffer. The concentration of the purified protein was measured using the Bradford protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA) with BSA as the standard.
4
Protein Purification via Chromatography
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Fast performance liquid chromatography was performed on a GE Healthcare Äkta Protein Purification System. Nickel affinity chromatography was performed with a HisTrap Excel 5-ml column (GE Healthcare). Gel filtration was performed by hand with disposable Sephadex® G-25 columns (GE Healthcare).
5
Engineered Nanobody-Fc Antibody Constructs
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The dimer constructs (DR14 and DS43) were engineered via fusing nanobody to hFc. The homo-trimer versions of R14 and S43 (TR14 and TS43) were constructed via head-to-tail with (GGGGS)3 linkers and a C-terminal His-tag. The coding sequence of R14 or S43 fused to the Fc of human IgM was cloned into the pCAGGS vector to generate IgM versions of the antibodies (MR14 and MS43). The coding sequence of TR14 was synthesized by Nanjing GenScript Biotech Co., Ltd., and TS43 was synthesized by Tsingke Biotechnology Co., Ltd. DR14 and DS43 were expressed in Freestyle 293F cells and were purified using a HiTrap Protein A 5-mL column (GE Healthcare) and Superdex 200 column (GE Healthcare). TR14 and TS43 were expressed in Freestyle 293F cells and were purified using a HisTrap EXCEL 5-mL column (GE Healthcare) and Superdex 200 column (GE Healthcare). The recombinant plasmid for MR14 or MS43 and a human J-chain expressing vector were co-transfected into Freestyle 293F cells to express the MR14 or MS43 proteins, which were further purified by HiTrap™ IgM Purification HP (GE Healthcare) and Superose 6 Increase 10/300 GL (GE Healthcare) chromatography.
6
Purification of Secreted UNC5B Variants
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Suspension-cultured HEK-293F cells transfected using either pHLSec-UNC5B-FL or pHLSec-UNC5B-Δ8 plasmids were harvested 6 days after transfection by 10 min centrifugation at 1,000 × g110 . The supernatant containing secreted UNC5B variants was loaded at 0.5 ml/min on a 1 ml HisTrap Excel 5 mL column (GE Healthcare). The column was washed with 10 column volumes of buffer A, composed of 50 mM HEPES, 500 mM NaCl, pH 8.0. Nonspecifically bound material was washed out by supplementing 35 mM imidazole to buffer A. Elution of the sample of interest was obtained by increasing the imidazole concentration to 250 mM. The elution peak protein was concentrated by centrifugation using Vivaspin® Turbo 15, 30,000 MWCO centrifugal filters (Sartorius) to a volume less than 500 μl and then loaded onto a Superdex 200 10/300 GL (GE Healthcare) gel filtration column equilibrated with 50 mM HEPES, 200 mM NaCl, pH 8.0. Both samples yielded single peak elutions corresponding to the UNC5B variants as assessed by SDS-PAGE analysis. Peak fractions were collected and further concentrated to 1 mg/ml. The purified samples were then flash-frozen in liquid nitrogen and stored at −80 °C until further usage.
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