Ni-column

[celina]

Hi everyone. This is the thing: i´m puryfying a protein with a histag with this system and it works fine, the only problem is another protein also interacts with the column and elutes together whith my protein! (The second protein does not have a his tag)
Any suggestions???
thanks again

[quatkorea]

Hi Celina,
seems to me that it is quite natural there are many very weak bands appearing together with a expected dark band in samples purifying by Ni NTA method when you run SDS-PAGE. The affinity could not be reached 100% purification and the scientists accept this. Depending on you purpose of using if you need absolute purification or not. If you would like to use a protein for observe the 3D-structure or sequencing, it should be complete purification. In these case, after His-tag purification, the samples are still contaminated other proteins, you should use further step as isoelectric precipitation and anion exchange chromatography.

[nandlalc]

yes you are right.other proteins bind with Ni.NTA because proteins may have sequence of his amino acids........so after passing through NiNTA can be used ion-exchange chromatography purification.. it will work to get 99% purity..

[celina]

thanks for the advice: the thing is i´m purifying a dimer and only one of the monomers carries the his-tag, but both of them stick to the matrix! Any way, I must try further purificaton steps...Bye bye...

[bwbrian]

Try using a cobalt column instead of a nickel column. It's well known that cobalt produces cleaner preps of his-tag proteins.