Precipitation Problems with Concentrating Proteins with PEG - (May/16/2012 )

I have been in the process of purifying some in-house made protein. I have been trying to concentrate what I have via PEG reverse dialysis. I've done this before without much problem, but lately I have been getting a lot of precipitate after concentration and dialysis. I've checked and this precipitate has the same MW of my protein so I believe my protein is precipitating out of solution. Is it because of the PEG. I am using PEG8000 and the dialysis tube I have is either 2-4.5 kDa or 12-14kDa. If you have some suggestions please help. Thanks

peg is used as a protein precipitant. as you reverse dialyze, peg gets solubilized and passes through the membrane into your protein solution. when the peg concentration inside the dialysis bag gets high enough the protein will precipitate. to avoid this you will have to limit the dialysis.

the good news is that peg precipitation of protein does not irreversibly denature the protein. you can resuspend the precipitate with fresh buffer.

you can try shortening the dialysis bag (making it tight, again) after you attain the volume reduction you want and dialyze against fresh buffer to reduce the peg concentration so that you can dissolve the precipitate.