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Purification: Difference between revisions
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(New page: ==Chromatography== ===Affinity Chromatography=== ====Immobilized Metal Ion Affinity Chromatography (IMAC)==== =====Methodology===== Immobilized metal ion affinity chromatography (IMAC...) |
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My protein precipitates following elution, what should I do? | My protein precipitates following elution, what should I do? | ||
*Use a different buffer system, e.g. Tris, HEPES, other Good buffers. Optimum binding occurs at pH 7.5-8.0. | *Use a different buffer system, e.g. Tris, HEPES, other Good buffers<ref>Good, N.E. et al. Hydrogen ion buffers for biological research. Biochemistry 5, 467-77 (1966).[http://dx.doi.org/10.1021/bi00866a011]</ref><ref>Ferguson, W.J. et al. Hydrogen ion buffers for biological research. Analytical biochemistry 104, 300-10 (1980).[http://dx.doi.org/10.1016/0003-2697%2880%2990079-2]</ref>. Optimum binding occurs at pH 7.5-8.0. | ||
*Following elution chelate any leached Ni<sup>2+</sup> ions using EDTA. | *Following elution chelate any leached Ni<sup>2+</sup> ions using EDTA. | ||
*Rapidly remove imidazole - dilution, desalting. | *Rapidly remove imidazole - dilution, desalting. | ||
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*L-arginine to reduce non-specific aggregation | *L-arginine to reduce non-specific aggregation | ||
*0.25-0.5 M trimethylaminoxide. | *0.25-0.5 M trimethylaminoxide. | ||
==Notes== | |||
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