Hello! Do you have some 10-20mM imidizole (for background) in your buffers? Did you treat with protease inhibitors? Maybe more column volumes of wash buffer too? If you are still having high background you could use a gradient of imidazole to elute. Make sure your solutions are clean filtered and free of proteases. (usually not the issue) Good luck!!

Hi,

I find that the best way to reduce non-specific binding for Ni-NTA is to reduce the amount of resin that I use. I typically use 0.5 mL resin per 1 L of culture as a starting point.

Matthew Edward Thornton Thanks a lot for your suggestions! With an expectation of a cleaner background for secreted protein I added only PEFABLOCK but no imidazole initially. I did 8-10CV wash with no imidazole buffer for the same reason. I could try the imidazole gradient if it improves the profile.

Ryan Wheeler Thanks a lot for your suggestion! Yes, I use the Ni-NTA resin volume based on overexpression level of each individual protein estimated in the pilot run.

Seema Nath Hello! I expect the addition of a background amount of Imidazole will help. If you are using a gradient pump try 50-300 mM Imidazole and collect fractions. If you are using a gravity column, you can still take fractions and test protein presence with adding a drop of eluate to a roughly equal size drop of Bradford reagent on a strip of parafilm. This procedure may be useful for your wash step, sometimes the concentrations are high and you really have to wash well. non-blue Bradford reagents indicates it is pretty clean. I hope it works out well.

Matthew Edward Thornton Thanks once again for the useful tips. Earlier I have used the Bradford step for assessing the washing and elution quality before running a gel but like many other methods this worked for some and not for every proteins I have worked so far. Taking your suggestions, I will proceed with a slightly modified plan and will keep you updated.