I treated Ni-NTA column with 10% APS because I purified a protein using a buffer containing DTT and it tourned brown. After APS treatment I wash the column with water and then I stripped it with EDTA, wash it again and finally used NiCl2 to recharge column, but when I washed again with water all the nickel was eluted. Is the column damaged?
Dithiothreitol should not be used with Ni-NTA because it reduces the nickel, resulting in the brown color. The column could be stripped with EDTA and recharged with nickel to be used again. However, it is important to remove the EDTA thoroughly because it will bind to the nickel.
What is APS? If it reacted chemically with the NTA resin, it would prevent further binding of nickel. The resin will have to be discarded.
Here my question is that why you using 10% APS? second thing is that i am totally agreed with Adam that DTT can chelate the NI-NTA resin.How much DTT you are using means the concentration you are using? Don't use APS for purification.
Adam and Guarav: APS(ammonium persulphate) is a way to remove reduced nickel. When it's brown and reduced it is not possible to elute it simply with EDTA.
It is quite possible that you damaged the resin. I am not sure but I've heard about using 1 or 0.1% APS to clean it. Make sure that you washed away all the EDTA before loading a column with Ni2+. If it didn't work the resin may be simply damaged.
Thank you for your answers. I think some how the APS damaged the resin, because I have washed very well the column with water and still Ni2+ does not bind. The concentration of APS I used was 1% and I leave it 30 minutes in the column, then wash it with water.
I also had the same issues of brown resin and also brown protein after elution and after a few online searches I came across this advice. A different step that you can try that I have used a few times now is if you want to use DTT (depending on concentration) you can wash the resin with your elution buffer first as the imidazole washes away any unbound free metal ions in the resin. Then re-equilibrate in lysis or wash buffer. From the limited experiments that I did the resin is not effected and you can then use it as normal with around 1-5mM DTT I think (would need to go back through my books to confirm). Higher concentrations may be detrimental so would require testing.
Roche have now released a resin, which by memory, is able to withstand 10mM DTT concentrations.
Tried to edit the last post but doesnt want to let me... The resin I was using was a Ni resin called PrepEase from USB with 1DTT and a 500mM Imidazole wash stopped the brown in the resin. If you made 500mM Imidazole in your brown protein elutions it also slowly turned back to clear. This comment was originally made based on GE Ni resin.
Dear Russell, thank you for your advise, but in this case using DTT is not the issue. I'm aware that I can use it just until 10 mM, usually I work with 5mM and even when my column turned brown it was working fine, but then I read that i can clean up the column with APS and I did that. Column was white again but when I tried to recharge with Ni2+ didn't bind it, that is why I'm wondering how APS could have damaged the resin. Any ideas?
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