Clontech Laboratories His60 Ni Superflow Cartridges Benutzerhandbuch - Seite 8

Blättern Sie online oder laden Sie pdf Benutzerhandbuch für Laborausrüstung Clontech Laboratories His60 Ni Superflow Cartridges herunter. Clontech Laboratories His60 Ni Superflow Cartridges 15 Seiten. Purification of expressed his-tagged proteins from bacterial, yeast, mammalian, and baculovirus-infected cells.

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His60 Ni Superflow Cartridges User Manual

C. Buffers – Denaturing Conditions – Guanidine-HCl or Urea

Denaturants, such as 5 M guanidine-HCl or 8 M urea, enhance protein solubility. Because overexpressed proteins

in prokaryotic systems are sometimes insoluble, you may need to purify proteins under denaturing conditions.

When using high concentrations of guanidine-HCl or urea, protein unfolding takes place. On-column refolding or

post-elution refolding is protein-dependent. When purifying proteins under denaturing conditions, we recommend

preparing buffers as indicated below.

Buffers with 6M Guanidine-HCl



Equilibration Buffer: 50 mM sodium phosphate, 6 M guanidine-HCl, 300 mM NaCl, 20 mM imidazole;

pH 7.4



Wash Buffer: 50 mM sodium phosphate, 6 M guanidine-HCl, 300 mM NaCl, 40 mM imidazole; pH 7.4



Elution Buffer: 50 mM sodium phosphate, 6 M guanidine-HCl, 300 mM NaCl, 300 mM imidazole; pH

7.4

Buffers with 8 M Urea



Equilibration Buffer: 50 mM sodium phosphate, 8 M urea, 300 mM NaCl, 20 mM imidazole; pH 7.4



Wash Buffer: 50 mM sodium phosphate, 8 M urea, 300 mM NaCl, 40 mM imidazole; pH 7.4



Elution Buffer: 50 mM sodium phosphate, 8 M urea, 300 mM NaCl, 300 mM imidazole; pH 7.4

NOTE: Samples containing guanidine-HCl cannot be analyzed by SDS-PAGE. A buffer exchange to a buffer

containing urea must be performed before SDS-PAGE analysis. Samples containing urea can be analyzed directly

by SDS-PAGE.

D. Enzymes



Benzonase (Sigma, Cat. No. E8263-5KU)



Recombinant DNase I (TaKaRa, Cat. No. 2270A)

E. Accessories for Automated Purification



A suitable liquid chromatography system (LC procedure only) with 1/16 inch tubing—or a binary pump

system (a quicker and more convenient alternative)



Additional connectors and fittings required to attach the cartridges to a Bio-Rad BioLogic or a classic

FPLC System (Section III.F)

F. Accessories for Syringe-Based Purification



Luer Lock Syringe Fittings (GE Healthcare, Cat. No. 18-1112-51) for syringe-based purification only



M6 FPLC Fittings (GE Healthcare, Cat. Nos. 18-1112-58 & 18-1112-57) for syringe-based or automated

purification

G. Optional



PD-10 desalting columns (GE Healthcare, Cat. No. 17-0851-01) to remove excess imidazole from the

final sample when required for downstream applications

PT5143-1

031716

www.clontech.com

Clontech Laboratories, Inc. A Takara Bio Company

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