Separation of glycerol from protein - (Dec/03/2013 )

Hi,

It could be a very stupid question, but just came in to my mind and thought of asking the best people in research . For storage of protein in -80, we add some % of glycerol in, so that the ice crystal could not destroy our protein. I was wondering, if it is possible to remove glycerol from protein by dialysis or some other method??

Then I thought people do sucrose gradient or glycerol gradient for various proteins separation. After visualizing on gel, do they separate protein from sucrose or glycerol?

I don't work with this sort of stuff, but you can use dialysis to remove these sorts of molecules.  You could probably also use some sort of gel filtration too.

The gradient for separation is usually based on density.   The components of the solution you have your protein in will depend on your end use.

Thanks bob . Same here, I also don't work on these stuffs. But I was thinking if people do that? I tried to google also but it dint help. Like in density gradient experiments, I've not seen people purifying the protein or that component from the fractions. 

Life would be so easy no, if this would be possible

Glycerol can be removed by dialysis or gel filtration.  I often buffer exchange material into or out of a 10% glycerol-containing buffer using small columns like Zeba's or PD-10's.

I've never used or heard of a glycerol gradient before but sucrose gradients are used all the time in virology to purify viruses.

The 10 kDa cutoff spin filters are easier for this application than dialysis, and let you do essentially arbitrary buffer exchange and concentration.

I agree with the ease of the spin filters.  Using them for buffer exchange is limited to protein solutions that are either dilute or that can handle be concentrated to the extent necessary to acheive 'buffer exchanged' status.  If you don't need concentration, the gel filtration columns are just as easy and more gentle on your protein.  It's good to identify how much buffer exchange is necessary for your application and then calculate how much buffer exchange you've acheived with either the spin filter or the gel filtration columns.  I've had to buffer exchange things twice in order to get efficient conjugation. 

Has anyone used PD-10 column for removing 70% glycerol from protein?