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Hi, I managed to significantly reduce the viscosity of the PEG solution via buffer exchange using a 100kDa MWCO ultrafiltration device. The following papers have fantastic tables of solutes with their hydrodynamic radii. Definitely worth a read, followed by printing and posting of the tables on walls next to the FPLC :) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055910/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304934/ Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org ---- Original message ---- >Date: Wed, 20 Aug 2014 18:57:07 +0000 >From: CCP4 bulletin board <[log in to unmask]> (on behalf of Alexander Aleshin <[log in to unmask]>) >Subject: Re: [ccp4bb] Removing PEG3350 >To: [log in to unmask] > > I meant application of GF as an ion exchange > column. > > Oh, my goodness! Ion exchange is something else! > It should read "buffer-exchange" = desalting column. > On Aug 20, 2014, at 11:48 AM, Alexander Aleshin > wrote: > > Dear Remie, > I meant application of GF as an ion exchange > column. You can use special ion exchange columns, > but our lab often uses preparative GF columns for > this task. We just load the column, keeping > sample volume < the void volume. Thus, we do not > concentrate a protein before an ion exchange, > only after it. But that is inevitable. When I am > afraid to loose a protein during its > concentrating, I concentrate shoulders of the > eluted peak first, then add a central part. > My point was that it might be okay to exchange > buffers by concentrating a protein, but other > molecules like Peg3K would not penetrate the > membrane as well as water or salts do, as a result > their reduction in concentration will be > unreliable. Like, you do a 10 fold > concentrating/delusion of a solution, but the > final concentration of PEG3K will drop only by 3 > fold... > Alex > On Aug 19, 2014, at 9:42 AM, Remie wrote: > > Hi Alex, > I disagree with you even though GF is always the > last step in my purifications. > Because it involves concentration before and > after the GF so during the concentration you can > already be doing the buffer exchange. > You use GF when you want to purify other protein > impurities if they are different sizes. Of > course it has other uses too. But not quite > practical for just changing buffer also > considering the amount of protein you could be > loosing along the process. If one is careful, > centripreps are best for concentrating and > changing the buffer. I tell you this from > experience with large hard to express proteins. > Best of luck, > Remie > On Aug 19, 2014, at 10:45 AM, Alexander Aleshin > <[log in to unmask]> wrote: > > Remie, > Actually, concentrating of a protein solution > is not the best approach to removing low MW > impurities, gel filtration chromatography is > more reliable and ... faster. > Regards, > Alex > On Aug 19, 2014, at 7:03 AM, Remie Fawaz-Touma > wrote: > > Hi Reza, I had to do this before. > This protocol works for any PEG and any > chemical to be removed from a solution: > buffer exchange into the new buffer you want > your protein to be in. There are ways to do > that by 15 mL Amicon concentrators from > millipore for large volumes, or if your > protein is already concentrated, there are > some small 0.5 mL concentrators from > millipore as well. > The key is to keep your spinning at low > speeds (concentrators manuals will tell you) > so you don’t precipitate or loose > your protein. Check your protein > concentration every 2 hours just to make > sure you are not loosing it on concentrator > surfaces and so on. > Good Luck, > Remie > On Aug 19, 2014, at 9:55 AM, Reza Khayat > <[log in to unmask]> wrote: > > Hi, > > Does anyone have a protocol for getting > rid of PEG3350 from a protein sample? > > Best wishes, > Reza > > Reza Khayat, PhD > Assistant Professor > The City College of New York > Department of Chemistry, MR-1135 > 160 Convent Avenue > New York, NY 10031 > Tel. (212) 650-6070 > www.khayatlab.org