Hi there,

I am purifying my protein with Ni-NETA column, but there are mutiple bands. Now I am trying ion-exchange column after Ni-NETA but surprising find out the peak is very very small -- the peak on Ni-NETA column is about 500mAU, while the peak on ion-exchange column is only ~50mAU. my protein is 40KDa and am using cenntrifugal filter device (10,000) to change the buffer, the instruction said recovery rate is about 90% and my supervisor said it is a very good one. I ran a gel and found out only few protein were lost during buffer exchange.

Is there anyone with such mutiple column experiences and could help me with this problem? your early help will be highly appreciated. thanks a lot for looking.

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I don't do that much protein purification, but I usually purify my His-tagged protein with Ni-NTA followed by FLPC purification.

If you have a strong signal follow Ni-NTA but not ion-exchange, is it possible that not all of the imidazole has been removed when you eluted from the Ni-NTA column, hence giving an high A280?

Just my tupence worth!

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you should not consider the height of the peak but rather the surface under the curve, the area.
If your peak is larger, then it's less high for the same amount of protein.

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Thanks, that is possible...I guess my imidazole is not pure enough so it has absorbance at 280..but at least my peak started from 0 to ~500...

Can u get a single band by Ni-NTA column? I tried different concentration of imidazole (20-45mM), tried to run the column twice,,but still have 4-5 bands...could you please let me know if you have any ideas to remove the unwanted bands? thanks a lot.......

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Thanks for your kind reply,,,yes, the height of peak is comparable with AUC (area under curve) because the volume is almost the same (~15ml)....do u have any suggestion to remove the unwanted bands after Ni-NTA column?? now i am trying ammonium sulfate precipitation before Ni-NTA column but not sure wheter it works....thanks a lot.

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If your protein is His-tagged you should be able to. We use a 12-His tag and use 60 mM imidazole to wash proteins with limited affinity for the column, then elute with 1 M imidazole, and just use a PD-10 column to remove the imidazole. Under these conditions I usually get a single band- if there are multiple bands, then they are very likely to be His-tagged, so are either proteolytic degradatation products or the result of aborted translation.

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thanks for your prompt reply...60mM imidazole looks high for me, my protein has 6-his tag and when I tried 30, 35, 40, 45mM of imidazole it still has multiple bands but yield decreased dramatically... I ran western blotting by his antibody, only 2 of the 4-5 bands are his-positive -- so i should be able to get rid of 2-3 unwanted bands but i couldn't...is 6-His tag too short? I used pET28 vetctor..thanks a lot.

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Well, I've never used a 6x His tag, so perhaps someone more knowledgeable could comment, but I would expect it shouldn't be significantly displaced by 45 mM imidazole - this is certainly the range in which His-rich proteins could be expected to elute from the column. If the yields of all of your bands decreases as you increase the imidazole concentration, it doesn't sound as if any of them are suitably His-tagged. Is it possible the conformation of your protein renders the His-tag inaccessible for column binding?

This article discusses contaminating protein during purification of 6x His-Tag protein with metal chelate affinity chromatography expressed in E. coli, maybe it will be of some use to you:

Hengen, P., “Purification of His-Tag fusion proteins from Escherichia coli.”, Trends Biochem Sci., 1995, 20(7), pp. 285-286

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thanks for your kind comments...i am thinking of add 12 his-tag....

thanks again for your kind help..

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OK, best of luck! Let me know if you get any joy with it!

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