I purified a nuclear receptor once and it requires high concentration of
salt (>500 mM) to remain soluble. Other osmolytes (glycerol, non-ionic
detergents) can replace salt for nuclear receptors (confirmed). I also heard
(although have not tried it) that macromolecular crowding agents (PEG20K,
ficol70) can also increase solubility. The point is, many intracellular
proteins, especially DNA-interacting ones stay stable in a "crowded"
environment (the cytosol and nucleoplasma are crowded).
The selection of crowding agents will depend on what experiments you will do
with your purified proteins.
----- Original Message -----
From: <proteins-request from oat.bio.indiana.edu>
To: <proteins from magpie.bio.indiana.edu>
Sent: Monday, January 21, 2008 2:03 AM
Subject: Proteins Digest, Vol 32, Issue 11
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>>> Today's Topics:
>> 1. Re: problem of concentrating protein (sandy_zhu_2006 from hotmail.com)
>> Message: 1
> Date: Sat, 19 Jan 2008 04:29:04 -0800 (PST)
> From: sandy_zhu_2006 from hotmail.com> Subject: [Protein-analysis] Re: problem of concentrating protein
> To: proteins from net.bio.net> Message-ID:
> <778fee5c-a2b5-4d01-83e5-1148e5baf1d2 from i29g2000prf.googlegroups.com>
> Content-Type: text/plain; charset=GB2312
>> On 1ÔÂ17ÈÕ, ÉÏÎç3Ê±18·Ö, "Dr Engelbert Buxbaum"
> <engelbert_buxb... from hotmail.com> wrote:
>> Am 10.01.2008, 01:01 Uhr, schrieb <sandy_zhu_2... from hotmail.com>:
>>>> > The protein I am purifying is a kind of zinc finger protein which
>> > has 81 amino acid and its theoretical pI is 9.19.But after purified
>> > with Hitrap Heparin column the target protein were still stable,then
>> > when I use the binding buffer(20mM Tris,10mM Mer,100mM NaCl and 1mM
>> > ZnCl2,pH 7.0)to dialyze in order to remove the 2M NaCl which were used
>> > in eluting the protein from the Heparin column, I found that there are
>> > many sediment in it.
>> > How to solve this problem?Is there any buffer which is more siutable
>> > than Tris? Since at pH7.0 Tris is not as efficient as it was.
>> > Plus I tried phosphate buffer,but it will interact with the ion
>> > zinc (within zinc finger protein) and produce sendiment too.
>>>> What is the composition of your elution buffer? Does it contain Zn? Does
>> the precipitation also occur when the binding buffer does not contain Zn?
>> My elution buffer do have Zn(1mM ZnCl2) in it.How to improve its
> is the mayor concern right now.
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