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> > 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 >> >>> 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 >>> ---- Original message ---- >>>> Date: Wed, 20 Aug 2014 18:57:07 +0000 >>>> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Alexander >>>> Aleshin <aales...@sanfordburnham.org>) >>>> Subject: Re: [ccp4bb] Removing PEG3350 >>>> To: 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> 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> 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 >
