> 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 <[log in to unmask]> 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 <[log in to unmask]<mailto:[log in to unmask]>> 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: [log in to unmask]<mailto:[log in to unmask]>
>>
>> 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 <[log in to unmask]<mailto:[log in to unmask]>> (on behalf of Alexander Aleshin <[log in to unmask]<mailto:[log in to unmask]>>)
>> Subject: Re: [ccp4bb] Removing PEG3350
>> To: [log in to unmask]<mailto:[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]<mailto:[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]<mailto:[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<http://www.khayatlab.org/>
>>
>>
>> <winmail.dat>
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