Recombinant FAB expression - purification (Feb/10/2011 )

Hello,

I am trying to express recombinant FAB. We have designed the vector so we just put a stop codon beginning at the the heavy chain constant region and co-transfect with the light chain. Expression is fantastic. Purification is another issue... We currently use anti-kappa columns from GE. This pull down the FAB, but also pulls down free light chain. It is important that I get this as pure as possible. The 25 kD difference does not seem to be enough to do a size exclusion. Anyone have any ideas that has done this before?

Jordan

you should be able to separate them on superdex 200. use a low sample volume to ensure sharp peaks and overlap will be minimized.