i think that there are 2 more probable causes:

protein degradation and lost of the his tag during the expression. you can check it performing a wb with anti-his on your soluble extract before ni-nta loading;

your protein aggregate as a multimer and also if the his tag is it present it in buried inside the multimer and not able to bind the ni-nta

what do you mean when you write thay the nickel is oxided?

ciao

Manuele

Manuele,

I have already checked both cell extracts containing the expressed protein and purified protein with anti-his; both were visualized just fine.

Instead of the Ni-NTA column being it's natural light blue color it changed to a brownish/orange. I suspect it may be due to high concentration of reducing agent in lysis buffer (I use 1mM DTT).

It could be then reduced not oxidized. Remove dtt to see that no color change occurs. although 1 mM should be fine. You can also pass the buffer through the column first.

Dear Thomas

i'm agree with Omar, if your coloumn become orange it means that there is s-ni bonds formed and sh of dtt compete with the binding of your protein. it is strange that it is due to 1mM dtt because the most resins are repoted to be stable up to 5mM vut the colour is quite clear.

if do you not use dtt in the lysis buffer is you protein not soluble? because some times protein with cysteines can be purified also with out dtt, it depends from the role and the surface exppsure of those cysteine

ciao

Manuele

To add above with omar and manuele, I wonder if you purchased protease inhibitor tablet/solution which is his compatible ? You might be seeing additive effect having DTT, along with the tablet issue - edta apart from this there is no way that you will find color change. It is very unlikely to have photo induced oxidation in such prep.

Hello Thomas, I agree with the others that 1 mM DTT should be OK even if not recommended. Check your DTT concentration, could you have miscalculated it? Does the brownish color disappear upon elution ? The reason why I'm asking is, protein binding can change the color of the resin but that's a reversible process: the resin returns to its blue color after elution. If not, it looks like the irreversible effect of sulfhydryls on Ni. You can also get a brownish color with very basic solutions (NaOH 1M for instance). So check your buffer for other sulfhydryl containing components and check the pH. About the absence of binding, low pH can be a problem, as well as high salt (especially chaotropic salts like KI or KBr) and detergents, so check for these too. In case the His-tag is masked as proposed by Manuele, you can:

-remove the imidazole from the wash as well and elute in steps : 30 mM 50 mM 100 mM imidazole, etc, because the affinity of your ptotein for Ni ions might just be reduced (the protein will be less pure but you can add subsequent purification steps)

-introduce a small linker between your tag and your protein (3-4 small aa like G, A or S)

-put your tag at the other end of the protein.

Olivier

Hi Thomas,

If the color change of your Ni-NTA resin is irreversible, it is recommended that you replace the column with a new one or regenerate the resin. In addition, it is recommended that you use 0.5 mM TCEP instead of DTT during the subsequent purification process.

All the best!

@Thomas: Although it is a little late, you don't need to replace the column after becoming reduced. Simply wash with acetic acid and continie with your regeneration protocol. You can find a suitable protocol here; it has been developed for Cube resins, but it should also work for your material. Just look for the regeneration protocol after DTT usage:

https://cube-biotech.com/media/pdf/08/34/42/Washing_Regenerating_HisAffinityResins.pdf

And as an alternative, you can use INDIGO Ni-Resin, which is stable against EDTA and DTT and doesn't lose binding capacity in presence of them:

https://cube-biotech.com/products/purification-resins/his-affinity-resins/indigo-ni-agarose-100/