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CCP4BB  August 2014

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Subject:

Re: Removing PEG3350

From:

Reza Khayat <[log in to unmask]>

Reply-To:

[log in to unmask]

Date:

Wed, 20 Aug 2014 16:18:49 -0400

Content-Type:

text/plain

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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 <[log in to unmask]> (on behalf of Alexander Aleshin <[log in to unmask]>)

>Subject: Re: [ccp4bb] Removing PEG3350  

>To: [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]> 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]> 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

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