glycerol is still there after dialysis - (Jun/15/2005 )

I have a new problem!! I have discovered that, even after dialysis of what appears to be purified protein, glycerol is attached itself to my protein. When we look at a raman spectrum, glycerol dominates. Although, I can not completely rid my buffers of glycerol during the purification process due to my protein precipitating. I need it to stay in solution and the glycerol is what does that. Why is the glycerol not leaving the dialysis membrane when I dialyze with ammonium bicarbonate?...or does any one have any suggestions to get the glycerol out?????

Dialysis removes solutes from the buffer and those molecules which adhere to the protein either lossely or reversibly. First of all, check if your dialysis is allright. Use a fresh activated memebrane and a minimum of 100 volumes of tank buffer. Change the tank buffer atleast twice.

If the glycerol persisites even after dialysis then the protein might be interaction with it. There are cases where glycerol used in the crytsallization buffer has been noted in the active sites of the proteins. In such cases it would be impossible to remove it.