Editing On Column Refolding (section)

== Purification and refolding ==

=== Loading and washing ===

Load the sample and wash the column with 10 ml 20 mM Tris-HCl, 0.5 M NaCl, '''5 mM''' imidazole, '''6 M guanidine hydrochloride''', 1 mM 2-mercaptoethanol pH 8.0. Change the buffer to 20 mM Tris-HCl, 0.5 M NaCl, '''20 mM''' imidazole,1 mM 2-mercaptoethanol, '''6 M urea''' pH 8.0 and wash with 10 ml.

=== Refolding ===

Refolding of the bound protein is performed by the use of a linear&nbsp; 6--> 0 M urea gradient, starting with the wash buffer above and finishing at one without urea. A gradient volume of 30 ml or higher and a flow rate of 0.1–1 ml/min can beused, while the optimal renaturation rate should be determined experimentally for each protein. Continue to wash with 5 ml of buffer without urea after the gradient has come to its endpoint.

=== Elution ===

Elute the refolded recombinant protein using a 10–20 ml linear gradient starting with 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0. and ending with the same buffer including 500 mM imidazole.

'''Binding Buffer''': 20 mM Tris-HCl, 0.5 M NaCl, '''5 mM imidazole''', '''6 M guanidine hydrochloride''', 1 mM 2-mercaptoethanol pH 8.0

'''Washing buffer''': 20 mM Tris-HCl, 0.5 M NaCl, '''20 mM imidazole''', '''6 M urea''', 1 mM 2-mercaptoethanol pH 8.0

'''Refolding buffer''': 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0

Refolding gradient: 30 ml

'''Elution Buffer''': 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0

Elution gradient: 10 ml

Fraction volumes: 3 ml sample loading, wash and refolding, 1 ml elution

Column: Ni2+-loaded HiTrap Chelating 1 ml

Sample: N-terminal (His) 6-tagged recombinant protein produced in E.coli

Flow rates: 0.1–1 ml/min, sample loading and refolding 1 ml/min, wash and elution
[[Category:Protocols]]