> We routinely use UFDF for buffer exchange. It always stroked me why such an advantageous technology is so expensive! How much a whole set of equipment costs?
Alex On Aug 20, 2014, at 3:48 PM, Lieh Yoon Low wrote: > 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>
