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His-tag purified protein is yellow! - (Nov/25/2008 )

Hello Folks,

I purified proteins (Cys-containing) under denaturing conditions (4-6M GuHCl, 100mM KPi) by pH elution on Ni-NTA beads. Recently I have been finding myself with yellowish proteins, and at one point when I neutralized the protein, the solution turned brown! (I have purified these proteins before and they do not have this discoloration - under the similar conditions.) This time I have been using regenerated Ni-NTA resin (following Qiagen's protocol and HPLC grade anhydrous ethanol). But not sure if the regeneration is the problem or something else - growth media? lack of reductant during purification? awful case of bad luck?

I do get quite a lot of yield. In addition to the yellowish color, the more pressing issue is that the proteins do not have thiol reactivity, hence I cannot use the Cys residues to add fluorescent labels.

So, may I know if anybody dealt with problems like this before? And how did you fix it? Any thoughts?

THANKS in advance!


Reduced Ni-NTA resin turns brown, also, reduced Ni can be stripped from column turning the eluting solution yellow.


I have the same problem, my resin turns bown/yellow and when I elute my eluates are brown/yellow while the resin turns back to bright blue.
This has to do with Ni ions being reduced or so by something in the buffer/media. Problem is that u cannot use Absorbance 280nm to check protein concentrations. I have tryed adding EDTA to the eluates to bind the nickel but the eluates still remain brown/yellow, I think its something in the media that binds to the resin and is eluted with the Imidazole and not the nickel.

Please give more detail, it could help solving, ex, what buffers u use? B-ME? DTT? do you purify peroteins from LB-media!?


The Novagen Ni-NTa manual has a reagents compability table; in short: high concentrations of Tris or other tertiary amines buffers, DTT, DTE, high concentrations of some amino acids (although it lists His as an alternative to imidazole, His can strip the column too).


Thanks for the thoughts...

I express my proteins in 2XYT media. I lyse my cells using 6M GuHCl, 100mM KPi pH 7.4. I do a batch elution using 4M GuHCl 50mM KPi pH 4. After elution, I make the proteins 2mM DTT and 1mM EDTA - in the hopes of keeping the protein under reducing conditions and removing any metal contaminants.

I ruled out the bead regeneration process, since I used new resin and the proteins are still yellow. Using urea-based buffers (8M urea, 100mM KPi, pH 4) appear to give clearer eluted proteins. I have no idea if it has something to do with the media or even the cells (I use BL21DE3 pLysS). I doubt it though since I have quite a good yield of proteins.

(Singel, as for using imidazole, isn't imidazole itself yellowish? I guess it depends on the concentration. Also, if you really want the protein concentration, could you run a gel? The BCA assay won't work either if your protein is still in imidazole. I hope you figure something out - and if so, please let me know! smile.gif )


Oh well, it's better not put EDTA in your sample buffer...

For the concentration measurement, could you try to remove imidazole first before further measurement? Like using some gel filtration column if you have large scale protein. if not, try some buffer exchange column from BIORAD, e.g. P6 or P30


There is Imidazol available that doesn't absorb at 280nm. Merck has one which is very pure, try getting that one, I never had a problem with it. I wonder if the reason PierceĀ“s BCA kit is not compatible with Imidazole is cause they have used other types of imidazole!? I will try it out.
I think the brown/yellow contaminants I see is from something in my Culture media (Peptone, Tryptone, Yeast Extracts).


QUOTE (Singel @ Dec 3 2008, 09:57 AM)
I think the brown/yellow contaminants I see is from something in my Culture media (Peptone, Tryptone, Yeast Extracts).

Just out of curiosity, can you see these potential media contaminants on a gel? If so, what molecular weight range do you think they are? I typically see degradation products for my proteins, so my gels are never really clean.