A S. denitrificans culture was grown in a final volume of 2 L of Marine broth pH 7.6 at 30 °C for 16 h. The culture was then harvested by centrifugation, resuspended in 0.02 M Tris-HCl buffer pH 8.0 and the cell suspension lysed with three passages through an EmulsiFlex (AVESTIN, Inc.) (10000 psi). The soluble fraction separated by centrifugation (186000 × g, for 20 min) was then applied to a High Q column (Bio-Scale Mini, UNOsphere Q Cartridge, 5 mL, BioRad) connected to an FPLC system (DuoFlow-BioRad, Hercules, USA). Protein elution was carried out with a linear gradient of NaCl (0–1 M) in 0.02 M Tris-HCl buffer pH 8.0 buffer at 2 mL/min. Fractions that were immunostained with the anti-shewasin D antibody (GenScript, Piscataway, USA) were pooled, diluted and applied to a Mono Q 5/50 (GE Healthcare Life Sciences) connected to an FPLC system (DuoFlow-BioRad, Hercules, USA). Protein elution was carried out with a linear gradient of NaCl (0–1 M) in 20 mM Tris-HCl buffer pH 8.0.
Mono q 5 50
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Mono Q 5/50 is a strong anion exchange chromatography column produced by GE Healthcare. It is designed for the purification and separation of charged biomolecules, such as proteins, enzymes, and nucleic acids. The column has a volume of 5 ml and an internal diameter of 5 mm.
Automatically generated - may contain errors
Lab products found in correlation
Ficin, by Merck Group (3 mentions)
Protein g, by GE Healthcare (3 mentions)
Ni nta, by GE Healthcare (2 mentions)
Ni sepharose 6 fast flow, by GE Healthcare (2 mentions)
Abspin trap protein g sepharose columns, by GE Healthcare (2 mentions)
Emulsiflex, by Avestin (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Matrigel, by Serva Electrophoresis (1 mentions)
Collagen r solution, by Serva Electrophoresis (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
3c protease, by GE Healthcare (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Mabselect sure, by Cytiva (1 mentions)
8 protocols using mono q 5 50
1
Purification of Shewasin D Protein
2
Purification and Expression of Antibody Fragments
3
Venom-Derived Biomaterial Synthesis
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Azocaesin, methylcellulose and fibrinogen were obtained from Sigma-Aldrich (St Louis, USA). Collagen R solution, Matrigel and basic fibroblast growth factor (bFGF) were obtained from SERVA electrophoresis (Heidelberg, Germany), BD biosciences (San Jose, USA) and R & D systems (Minneapolis, USA) respectively. The crude venom samples of B. moojeni and B. atrox were purchased from San Maru Serpentarium, Brazil, and their use was approved by the Brazilian Institute of the Environment andef Renewable Natural Resources (Ibama). Purification columns Superdex 200 column and Mono Q 5/50 were purchased from GE Healthcare (Little Chalfont, UK).
4
Production and Purification of Monovalent Streptavidin
5
Purification of IgG mAbs and Fabs
6
Purification and Antibody Production of CD0386
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Following nickel affinity purification as described for SrtB, recombinant CD0386 was dialysed into 20 mM Bis-Tris pH 5.5, 180 mM NaCl and applied at a rate of 1 ml/min to an equilibrated MonoQ 5/50 anion exchange column (GE Healthcare). The column was washed until absorbance at 280 nm returned to the baseline level, at which point a gradient was initiated to 260 mM NaCl over 30 CV. Fractions containing CD0386 were dialysed into a storage buffer consisting 25 mM HEPES, 150 mM NaCl. Production of rabbit antisera was performed by Covalab S.A.S. A 50 μg dose of either SrtB or CD0386 was mixed with Freund's incomplete adjuvant and injected subcutaneously into duplicate New Zealand White rabbits at 0, 21 and 42 days. A terminal bleed was performed on day 53, and sera were assayed by ELISA with recombinant CD0386.
7
Purification and Expression of Antibody Fragments
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Igs were purified from 200μl of mouse or macaque serum using Ab
Spin Trap protein G Sepharose columns (GE Healthcare, #28–4083-47).
Ig-containing fractions were buffer exchanged with PBS by overnight dialysis at
4ºC (dialysis cassettes 20000 MWCO Thermo Scientific, #66005).
For structural studies, mouse IgGs and macaque His6-tagged
Fabs were expressed by transient transfection in HEK293–6E or Expi293
cells and purified from cell supernatants using protein A or G (GE Healthcare)
(for IgGs) or Ni-NTA (GE Healthcare) or Ni Sepharose 6 Fast Flow (GE Healthcare)
(for Fabs) chromatography and SEC44 (link). Mouse Fab was obtained by digesting IgG at 1–5 mg
ml−1 with ficin (Sigma). Fab was purified by protein G (GE
Healthcare) and SEC chromatography45 (link), followed by monoQ 5/50 (GE Healthcare) ion exchange
chromatography. The common iGL of the PGT121 and 10–1074 bNAbs17 (link) was expressed as a
His6-tagged Fab.
Spin Trap protein G Sepharose columns (GE Healthcare, #28–4083-47).
Ig-containing fractions were buffer exchanged with PBS by overnight dialysis at
4ºC (dialysis cassettes 20000 MWCO Thermo Scientific, #66005).
For structural studies, mouse IgGs and macaque His6-tagged
Fabs were expressed by transient transfection in HEK293–6E or Expi293
cells and purified from cell supernatants using protein A or G (GE Healthcare)
(for IgGs) or Ni-NTA (GE Healthcare) or Ni Sepharose 6 Fast Flow (GE Healthcare)
(for Fabs) chromatography and SEC44 (link). Mouse Fab was obtained by digesting IgG at 1–5 mg
ml−1 with ficin (Sigma). Fab was purified by protein G (GE
Healthcare) and SEC chromatography45 (link), followed by monoQ 5/50 (GE Healthcare) ion exchange
chromatography. The common iGL of the PGT121 and 10–1074 bNAbs17 (link) was expressed as a
His6-tagged Fab.
8
Nucleosome Reconstitution and Purification
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The DNA and the histone octamer complex were mixed in a 1:1.5 molar ratio in the presence of 2 M KCl. Reconstitution of the H2A–H2B(T122C–biotin)–H3.1–H4 complex was performed by incubating the components at a 1:1.5:3 molar ratio (DNA:H2A–H2B:H3.1–H4). The samples were dialysed against refolding buffer (RB) high (10 mM Tris-HCl pH 7.5, 2 M KCl, 1 mM EDTA and 1 mM DTT). The KCl concentration was gradually reduced from 2 M to 0.25 M using a peristaltic pump with RB low (10 mM Tris-HCl (pH 7.5), 250 mM KCl, 1 mM EDTA and 1 mM DTT) at 4 °C. The reconstituted nucleosomes were incubated at 55 °C (or 37 °C in the case of LIN28-E and Por endogenous nucleosome sequences) for 2 h followed by purification on a Mono Q 5/50 ion-exchange gradient (GE Healthcare), and dialysed into 20 mM Tris-HCl pH 7.5 and 500 μM TCEP overnight. Nucleosomes were concentrated and stored at 4 °C.
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