Coenzyme A (2.5 mM) was added to a solution of ACP (0.8 – 1.0 mM) with Sfp R4–429 (link) (1.5 μM) in 50 mM sodium phosphate, 10 mM MgCl2, pH 7.6, for a total volume of 1 mL. The solution was incubated for 12 hrs at room temperature. The reaction mixture was desalted into 50 mM phosphate buffer, pH 7.6 using a HiPrep 26/10 column (GE). Percent conversion to holo-ACP was determined using liquid chromatography mass spectrometry (LCMS).
Hiprep 26 10 column
Manufactured by GE Healthcare
Sourced in United States
The HiPrep 26/10 column is a size exclusion chromatography column designed for the separation and purification of biomolecules. It features a 26 mm diameter and 100 mm bed height, and is compatible with a variety of ÄKTA chromatography systems.
Automatically generated - may contain errors
Lab products found in correlation
Hiprep, by Cytiva (10 mentions)
Histrap, by GE Healthcare (2 mentions)
Q sepharose, by GE Healthcare (2 mentions)
Histrap excel column, by GE Healthcare (2 mentions)
Nanodrop lite, by Thermo Fisher Scientific (1 mentions)
Mabselect, by Cytiva (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Beyotime (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Merck Group (1 mentions)
Transb de3 competent cells, by Transgene (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Sp sepharose column, by GE Healthcare (1 mentions)
Akta purifier, by GE Healthcare (1 mentions)
Thrombin, by Roche (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Ultrafree mc centrifugal filter unit, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Apoferritin, by Merck Group (1 mentions)
Thyroglobulin, by Merck Group (1 mentions)
12 protocols using hiprep 26 10 column
1
ACP Holo-Conversion by Sfp Enzyme
2
Determination of Ligand-Kinase Crystal Structures
3
Purification of cB72.3 IgG4 Antibody
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The CCS containing B72.3 IgG4 was allowed to thaw on ice, centrifuged at 4601g for 10 minutes and then passed through a 0.45 μm Supor Acrodisk syringe filter to remove large particulate matter. The filtered media was desalted using a Hiprep 26/10 column (GE Healthcare Life Sciences, Little Chalfont, UK) in equilibration buffer (50 mM phosphate/150 mM NaCl, pH 7.4). The desalted media was loaded onto a Protein A MabSelect SuRe column (Cytiva, UK) equilibrated with equilibration buffer. The column was washed with equilibration buffer and bound material eluted with 0.1 M sodium citrate, pH 3.3. The purified IgG4 was eluted into neutralization buffer (1 M Tris-HCl, pH 9.0) to raise the pH. The fractions containing the mAb were pooled and the purified cB72.3 IgG4 fractions were buffer exchanged into equilibration buffer and concentrated using 100 kDa molecular weight cut off filters (Merck Millipore, Billerica, MA, USA). The protein was concentrated to 10 mg ml−1. The amount of cB72.3 IgG4 was quantified by OD280 nm using a Nano drop lite (Thermo, Wilmington, DE, USA) with an E1% of 13.7. Samples were stored at −80 °C for further use.
4
Protein Purification and Ligand-Kinase Complex Formation
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Experiments
involving target–drug complex formation, data collection, and
data processing were done using the standard operating protocol as
validated previously6 (link) for the determination
of atomic resolution ligand–kinase crystal structures. Protein
purification6 (link) was done using a combination
of affinity-based adsorption chromatography (HisTrap; GE Healthcare),
gel filtration (HiPrep 26/10 column; GE Healthcare), and ion exchange
chromatography (Q Sepharose; GE Healthcare).
involving target–drug complex formation, data collection, and
data processing were done using the standard operating protocol as
validated previously6 (link) for the determination
of atomic resolution ligand–kinase crystal structures. Protein
purification6 (link) was done using a combination
of affinity-based adsorption chromatography (HisTrap; GE Healthcare),
gel filtration (HiPrep 26/10 column; GE Healthcare), and ion exchange
chromatography (Q Sepharose; GE Healthcare).
5
Purification of Bispecific Antibodies
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Both His-tagged BsAbs were purified using immobilized metal affinity chromatography with a 5 mL HisTrap excel column (GE Healthcare). The column was equilibrated in 20 mM sodium phosphate pH 7.4, 500 mM NaCl. The harvested supernatant was loaded onto the column and then washed with equilibration buffer containing 20 mM imidazole to remove non-specifically bound contaminants. The protein of interest was then eluted with 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 500 mM imidazole. The final product was desalted into PBS using HiPrep 26/10 column (GE Healthcare) and then filtered through a 0.22 μm syringe filter. The concentration of protein was determined by measuring the UV absorbance at 280 nm using a Nanodrop 1000 spectrophotometer. Protein purity was analysed by SDS-PAGE using 4–12% Bis-Tris gels (Invitrogen).
6
Purification of Strep-tagged Proteins
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Cell pellets were resuspended in 3 ml of 50 mm Tris-HCl (pH 7.9), 20 mm EDTA, 0.2 mm phenylmethanesulfonyl fluoride, 2 μm pepstatin, 2 μg/ml DNase for each gram of cells (wet weight), and cell-free extracts were prepared as described above. The cell-free extract was applied to a 5-ml prepacked Strep-Tactin®XT Superflow® gravity flow column (IBA Lifescience) equilibrated with 50 mm Tris-HCl (pH 7.9), 20 mm EDTA (buffer B). The loaded column was washed with buffer B until the eluting fractions exhibited no absorbance at 280 nm . Column-bound proteins were eluted using buffer A having 50 mm biotin and subsequently desalted using a HiPrep 26/10 column (GE Healthcare) equilibrated with buffer B. Samples were frozen at −80 °C until used.
7
Purification and Characterization of LBD1
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The recombinant pCold-SUMO-LBD1 plasmid was transformed into TransB (DE3) competent cells (TransGen Biotech, China) to induce the expression of His6-SUMO-LBD1 with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 18 h at 16°C in the presence of 0.5 mM CaCl2 in LB medium. His6-SUMO-LBD1 was purified using prepacked Ni-NTA affinity column (GE Healthcare, USA). Phenylmethanesulfonyl fluoride (PMSF) (Sigma, USA) was applied during the entire purification process to inhibit the protease activity. Purified His6-SUMO-LBD1 was subsequently desalted with HiPrep 26/10 column (GE Healthcare, USA) to remove extra imidazole. His6-SUMO tag was separated from LBD1 by Ni-NTA column followed the treatment of SUMO protease (20 U/μL) at 4°C for 16 h. Purified LBD1 was analyzed by 12.5% SDS-PAGE and Superdex 200 (GE Healthcare, USA) to determine its molecular weight and conformation in solution. LBD1 was concentrated via ultrafiltration and its final concentration was determined by BCA kit (Beyotime, China). The truncated mutants were expressed and purified as a similar purification procedure of the wild type LBD1 except His6-Trx tag of pET-32M∙3C vector was cleaved from LBD1 mutants by PreScission protease (GE Healthcare, USA).
8
Purification of Cellulase Enzymes EG VI, CBH IIa, and CBH IIb
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A 20 ml sample of the whole cocktail obtained with the procedure described above was loaded onto an SP-Sepharose column (GE Healthcare) equilibrated with the same buffer (50 mM sodium acetate, pH 5.0). EG VI was eluted with 1 M sodium chloride, 50 mM sodium acetate buffer (pH 5.0). Collected fractions were desalted using HiPrep 26/10 column (GE Healthcare) and were analyzed by mass spectrometry and SDS-PAGE to identify and check their purity. CBH IIa and CBH IIb were purified following the procedures described by Bukhtojarov et al. [35 (link)] and Gusakov et al. [27 (link)] respectively.
9
Oligonucleotide Synthesis and Purification
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DNA oligonucleotides were synthesized on solid support using a H-8 DNA synthesizer (K&A Laborgeraete). Oligonucleotides were removed from solid support using ultra fast treatment with 50% ammonium hydroxide/50% methyl amine (AMA). Incubation with AMA took place for 20 min at room temperature followed by 15 min at 65°C. The solution was evaporated and immediately redissolved in 10 mM TEAB buffer. Oligonucleotides were purified with reverse phase HPLC on a C18 column. The mobile phase was evaporated and the remaining DNA was treated for 20 min with 80% acetic acid to remove the DMT group. Subsequently, the DNA was precipitated with ethanol and again redissolved in TEAB buffer. A GE AktaPurifier with a HiPrep 26/10 column was used to desalt the DNA. After the final evaporation, the DNA was dissolved in the NMR buffer containing 10% 2H2O, 20 mM K phosphate buffer (pH 7) and 80 mM KCl. Final concentration of oligonucleotides was in the range between 0.5 and 1.0 mM.
10
Purification of Nus-tagged Proteins
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