protein purification Ni-NTA

[piefke]

Hi

I´ve one little question. I purified an enzyme with Ni-NTA-Coloumns. And it worked, but I always have some bigger proteins running a little bit above my target-protein on the gel. I now have to explain this in my diploma thesis. I would understand if there would always be some smaller ones. then I would say these are truncated forms of the tagged protein. but these ones are larger...Do you have some ideas?

[blcr11]

Most likely they are non-specifically bound contaminants. That happens regularly with NTA columns. If they are, they should go away after further purification steps. Another possibility is that they co-purify with your target because they bind to the target--which is potentially interesting depending on what they are. If the extra proteins are binding to your target, doing things like changing the ionic strength of the buffer might change the amount or number of extra bands you see on the gel, or even remove them altogether if high or low ionic strength prevents binding completely. You can also try different IMAC resins to see if there are differences in the banding patterns, again suggesting that they could be non-specifically bound contaminants.