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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 09:08, 3 October 2007
, 3 October 2007→Notes
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*Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O you start with (maybe 50% of final volume) | *Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O you start with (maybe 50% of final volume) | ||
*Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column. Perhaps supplement the other buffers as well? | *Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column. Perhaps supplement the other buffers as well? | ||
*Leprevost adjusted the Imidazol concentration in Washing and Elution buffers. A high Imidazol concentration is needed in order to elute the 6xHis-tagged protein. | |||
*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. | ||
