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
