Tat-protein purification by Ni-NTA affinity

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Tat-protein purification by Ni-NTA affinity

Post by alondra06 » Sat Jan 02, 2010 2:23 am

I am purifying a TAT- protein and after the Ni-NTA affinity and the extra PD-10 desalting column I obtained an extra peak at 230 nm, narrow, well defined that reached 3.4 units of absorbance, measured by Nanodrop A280nm program. I found that it could be carbohydrates (which is logical considering where the protein came from), EDTA, phenol, and Guanidine HCL. I used GuHCl at 6M to disolve the bacterial harvest. Do you have any experience with it? I haven't done the western to check the protein but it worked once but I need to remove this contaminant before treating the cells with the solution. Please, any recommendation would be very welcome.
Thanks in advance

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Post by JackBean » Sat Jan 02, 2010 11:59 am

If you used the desalting column, you should not have any low-molecular-weight substances ;)So, check your column, if is it working properly, if it is, than you have probably some sugars, but I don't know, why should they be retained on Ni-NTA.
Of course, you should consider everything, what you have in your buffer ;)

Cis or trans? That's what matters.

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