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Knight:Purification of His-tagged proteins/Denaturing: Difference between revisions
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Knight:Purification of His-tagged proteins/Denaturing (view source)
Revision as of 15:42, 17 December 2006
, 17 December 2006no edit summary
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*10 mM imidazole (recommended by Kathleen, 9/27/2006) | *10 mM imidazole (recommended by Kathleen, 9/27/2006) | ||
*pH 8.0 | *pH 8.0 | ||
*1 mM TCEP (add just before use | *1 mM TCEP (add just before use) | ||
===Wash buffer (Qiagen buffer C)=== | ===Wash buffer (Qiagen buffer C)=== | ||
| Line 18: | Line 18: | ||
*10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?) | *10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?) | ||
*pH 6.3 | *pH 6.3 | ||
*10 mM TCEP (add just before use | *10 mM TCEP (add just before use) | ||
===Elution buffer (Qiagen buffer E)=== | ===Elution buffer (Qiagen buffer E)=== | ||
| Line 26: | Line 26: | ||
*10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?) | *10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?) | ||
*pH 4.5 | *pH 4.5 | ||
*1 mM TCEP (add just before use | *1 mM TCEP (add just before use) | ||
===Notes=== | ===Notes=== | ||
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*The urea should be freshly prepared and deionized prior to use. | *The urea should be freshly prepared and deionized prior to use. | ||
*The buffers should each be degassed before use. | *The buffers should each be degassed before use. | ||
*[http://www.piercenet.com/Products/Browse.cfm?fldID=02051012 TCEP] is not particularly stable in phosphate buffers, especially at neutral pH. Therefore, if TCEP is to be used in phosphate buffers, prepare the working solution immediately before use. | |||
==Procedure== | ==Procedure== | ||
