Alex, We routinely use UFDF for buffer exchange, instead of concentrating, we do DF, and usually 7 diavolume is enough to complete the process. If you have a right membrane cut off and a right equipment, DF is actually a very efficient way to do buffer exchange. The smallest volume we do, using Millipore equipment is few hundred ml, not sure if there is anything that can handle a few ml volume.
Ray Lieh Yoon Low > On Aug 20, 2014, at 6:38 PM, Alexander Aleshin <aales...@sanfordburnham.org> > wrote: > > I agree with Alex and Roger. > But my mentioning of plates number for a concentrator was an act of > stupidity! I bet it is possible to introduce such a concept for > concentrators, but it will strongly depend on a ratio of a molecule and a > pore sizes and a degree of concentration. In other words, efficiency of > molecules separation by a concentrator is hard to describe quantitatively. > > On Aug 20, 2014, at 3:04 PM, Lieh Yoon Low wrote: > > I agree with Alex and Roger. Just a matter of choosing the right SEC column. > > Ray > > Lieh Yoon Low > > On Aug 20, 2014, at 4:56 PM, Alexander Aleshin > <aales...@sanfordburnham.org<mailto:aales...@sanfordburnham.org>> wrote: > > The efficiency of a size exclusion column is proportional to the number of > theoretical plates (plate number). > I would say that a concentrator has plates number=1, while any preparative > column would have it >1, so SEC would always separate two different size > objects better. > > > On Aug 20, 2014, at 1:44 PM, Roger Rowlett wrote: > > Excellent references. PEG 3350 appears to be hydrodynamically equivalent to a > 20 kD globular protein. So for efficient separation, your protein needs to be > significantly larger than 20 kDa on a GEC column. In a centrifugal filter > (which is very inefficient--you need many exchanges and dilutions with buffer > to get nearly quantitative removal) it is possible that "snaking" of linear > polymer molecules through the pores might contribute to slightly more > efficient removal than expected based solely on hydrodynamic radius. > > GEC or a desalting column is definitely the quickest way to do this, if > possible. Flow rates may have to be slow (hence a typical flow rate column > separation) to allow for efficient distribution of solutes in the sample > solution if it has increased viscosity. > > Cheers, > > _______________________________________ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu> > > On 8/20/2014 4:18 PM, Reza Khayat wrote: > 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<http://www.khayatlab.org/> > > > ---- Original message ---- > Date: Wed, 20 Aug 2014 18:57:07 +0000 > From: CCP4 bulletin board > <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> (on behalf of Alexander > Aleshin <aales...@sanfordburnham.org<mailto:aales...@sanfordburnham.org>>) > Subject: Re: [ccp4bb] Removing PEG3350 > To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> > > 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 > <aales...@sanfordburnham.org<mailto:aales...@sanfordburnham.org>> 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 > <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> 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<http://www.khayatlab.org/> > > > <winmail.dat>
