Hi, There is a gray area, where you would refrain from talking about low-MW contaminants. PEG3350 will be a highly elongated polymer, with way higher hydrodynamic radius than a globular molecule of the same MW. It also will bind a lot of H2O. Hence, it might not go through concentrators (but could actually get concentrated), and also could still be present in the protein fractions after GF.
But I might be wrong, of course. Petri On 20 Aug 2014, at 20:48, Alexander Aleshin <[email protected]> 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 <[email protected]> > 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 <[email protected]> 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 > > >
