Hi all,
At the beginning, I was using native His-tag purification beads to purify my proteins; however, it still has proteins binding on bears, poorly eluted but can still see faint bands on western blot. Then, I am thinking to try denaturing conditions to purify His-tag proteins. When I did western blot, no bands are detected or visible. What are possible reasons for that? Is that due to boiling of my denaturing samples?
To perform purification by Ni affinity chromatography under denaturing conditions, the sample and column buffers can contain 6 M urea or guanidine-HCl. This will denature the protein and will not interfere with the chromatography. Boiling the protein will denature it, but it will probably also precipitate unless kept in solution by detergent such as SDS, so using urea or guanidine is preferable to boiling.
Be aware your protein will be carbamylated after urea and heat simultaneous treatment. What was the initial elution strategy such as imidazole conc.? Have you ever tested the EDTA elution before passing it over to denatured elution?
If you would like to avoid denaturing your protein, you can try moving the His tag to the opposite side of the protein (C-terminus to N-terminus, for example).
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