I performed a Histrap IMAC purification to purify a His-tagged archaeal TF after an overexpression protocol.
I did this experiment two times for the same TF.
I only slightly altered the overexpression protocol during the second experiment (adding DNaseI treatment and heat treatment steps). But the purificaton protocol remained exactly the same (same HisTrap colomn, same AKTA, same buffers, ...)
The elution profile of both purifications look different. The first one shows a clear peak around an elution volume of 30 mL, the second one shows a subtle peak around elution volume of 55 mL.
SDS-PAGE indicates that both experiments were succesfull, showing only the wanted TF in the fractions around the peaks. But I wonder: how can it be that the elution profiles were so different?
Is it really the same? Did you replace metal ions with fresh ones?
Actually, if you look carefully, even in the first round you had this wide, low peak .
First of all, changing 2 parameters (including heat treatment step) is not "slightly" different, so a different outcome is not surprising. One possible explanation could be aggregation/oligomerization of the target protein in which case you would have multiple tags on one aggregate, leading to a delayed elution at higher imidazole concentration. As stated before, there is also a low peak in that range in the first run. You wouldn't see any difference in SDS-PAGE but maybe you could compare these fractions by gel filtration.
Without seeing the complete chromatogram and your method it is difficult to say what happened. An unexpected peak in the very beginning of the gradient could be caused by a programming error where the is a pump wash for the B pump alone. In that case you get a brief burst of almost 100% B. And as others have mentioned your treatments of the sample were different, so that might be the reason.
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