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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