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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 08:31, 25 August 2006
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==Overview== | ==Overview== | ||
"The high affinity of the Ni-NTA resins for 6xHis-tagged proteins or peptides is due to both the specificity of the interaction between histidine residues and immobilized nickel ions and to the strength with which these ions are held to the NTA resin." <cite>QiagenNTAManual</cite> | "The high affinity of the Ni-NTA resins for 6xHis-tagged proteins or peptides is due to both the specificity of the interaction between histidine residues and immobilized nickel ions and to the strength with which these ions are held to the NTA resin." <cite>QiagenNTAManual</cite> | ||
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==Notes== | ==Notes== | ||
*Sauer lab uses a Qiagen Ni-NTA resin but | *Sauer lab uses a Qiagen Ni-NTA resin but this protocol uses spin columns. (Smaller scale purification). | ||
*Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields. | *Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields. | ||
*A lower pH may be needed for elution. For instance, aggregates should elute at pH 4.5 whereas monomers generally elute at pH 5.9. | *A lower pH may be needed for elution. For instance, aggregates should elute at pH 4.5 whereas monomers generally elute at pH 5.9. | ||
*Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column. | *Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column. | ||
*20 year old spin columns don't work. :) | |||
==Safety== | ==Safety== | ||
