[SoSki1620]

Hello,

I am looking for a protocol for a buffer and detergent exchange.  I work with F1Fo ATP Synthase from S. cerevisiae normally in a Bicine buffer with some various salts and extracted with DDM at a [Final] of 0.05%.  I am looking to do some experiment with the enzyme from e. Coli and received some enzyme from another university.

The buffer they use is a MES/Tricine buffer with 1% octyl glucoside and other salts .  I would like to exchange the buffer with mine and also exchange the detergent.  Since OG is able to be dialyzed I could remove it this way; the problem I have is that removal of the detergent will cause the enzyme to precipitate in the dialysis membrane.

I could though add DDM to a [Final]=0.05% directly to the enzyme and then dialyze it in my buffer.  However, will I then have mixed micelles with both OG and DDM or two separate micelles with the OG being dialyzed out and DDM remaining.

Another way I am thinking is to do the same thing in regards to the detergent but have my dialysis buffer be the same as theirs.  Then after the detergent is removed, pass it though a spin column equilibrated with my buffer.

A third way I am looking at is to remove the detergent using BioRad Bio-beads SM-2 Absorbants.  This would result in my enzyme likely precipitating in the tube but I can then redissolve it in my buffer.

I know a easy answer would be to try all these and see what works but I only have a limited about of enzyme to work with and can't keep having them send me more.

Thanks in advance for the advice.