4 replies to this topic

[badscientist]

Hi Folks,
I am trying to purify a protein with 3 Hisx6 tags. Any ideas on how high I can go with imidazole in the washes without losing my protein? I am getting loads of background even under stringent conditions (6M Guanidine HCl or 8M Urea). I think the trouble is that I am purifying from yeast and yeast has a lot of histidine rich proteins. Any ideas or suggestions?
Thanks.

[mdfenko]

you can try a gradient to see where your protein starts to elute.

or

you can wash with steps of increasing imidazole. initially, you can use larger steps then use smaller steps in subsequent runs until you find the optimal wash and elution concentrations.

[badscientist]

Thanks! Any suggestions on what range I should start with?

[mdfenko]

according to the book, "affinity chromatography handbook", from ge healthcare, elution is routinely performed with 500mM imidazole. they show a specific chromatogram where they equilibrate and wash with 100mM imidazole and elute with 500mM.

you may want to start with 50 or 100mM steps and refine from there.

Attached Files

•  affinity chromatography.pdf   2.82MB   97 downloads

[badscientist]

Thanks a million!