Sauer:Purification of His-tagged proteins/Denaturing prep: Difference between revisions
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This is a general protocol for purification of His<sub>6</sub>-tagged proteins under denaturing conditions. Note that you may need to modify this protocol to fit your specific needs. | =Introduction= | ||
This is a general protocol for purification of His<sub>6</sub>-tagged proteins under denaturing conditions. Note that you may need to modify this protocol to fit your specific needs. This protocol is a modified version of that provided with Ni<sup>2+</sup>-NTA resin from [http://www1.qiagen.com/SelectCountry.aspx Qiagen]. | |||
=Buffers= | =Buffers= | ||
Revision as of 14:51, 8 February 2006
Introduction
This is a general protocol for purification of His6-tagged proteins under denaturing conditions. Note that you may need to modify this protocol to fit your specific needs. This protocol is a modified version of that provided with Ni2+-NTA resin from Qiagen.
Buffers
Lysis buffer
100 mM NaH2PO4
10 mM Tris base
6 M guanidine hydrochloride
10 mM imidazole
Adjust to pH 8
Wash buffer 1
100 mM NaH2PO4
150 mM NaCl
8 M urea
20 mM imidazole
Adjust to pH 8
Wash buffer 2
50 mM NaH2PO4
500 mM NaCl
20 mM imidazole
Adjust to pH 8
Elution buffer
50 mM NaH2PO4
500 mM NaCl
250 mM imidazole
Adjust to pH 8
