Purifying His-Tagged Proteins from Insects cells on IMAC resin? - (May/01/2007 )

Hi,

Anyone as suggestion on the best way to purify His-Tagged proteins produced in Insect cells on a Ni+resin?

It appears that the Insect Cell culture Media (all the one we tested) is stripping the Ni+ from the column. I was told that filtration might be necessary before I can load on the Ni+ column... I'd like to avoid that as we don't have a filtration system.

Any other step I could do to help load my 5-10L of media containing His-tag proteins on the Ni+ column?

What about Ion Exchange? Any suggestion would be appreciated?

hi,
dialyze your insect media into the binding buffer over a night or 2 (just make sure you change the media about 4-6 times). in theory you should dialyze against 10X your volume, so seeing you have 5-10L you might need to do this in separate batches.


good luck!