- Protein crystallography
Main steps: - Protein purification
- Crystallisation
Special: - Programs for crystallography
- X-ray detectors
Basic tutorials: - Chemistry
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Xtal community: - CCP4BB
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Subject: Re: how to purify protein in its native form
From: Roger Rowlett rrowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2009-04-15
The holy trinity of protein chromatography is ion
exchange (IEX), hydrophobic interaction chromatography (HIC), and gel
exclusion (GEC), assuming no affinity purification is possible.
Depending on the abundance of protein, these three steps, perhaps in
concert with additional chemical steps, including but not limited to
salt precipitation and/or thermal denaturation, are often sufficient to
purify proteins to homogeneity from natural sources. For minisculely
abundant proteins, purification can be nightmarish. (Been there done
that, got the dirty T-shirt.)
The basis for any protein purification is an efficient assay for
determining the presence of the protein in a mixture, even at low
concentration. If you don't have a good assay (activity, antibody
binding, spectroscopic ligand, etc., you are in dire straits indeed.
But if you have an assay, you may not need to purify your protein at
all to determine its oligomerization state in vivo. You may only need
to pass a suitably concentrated crude extract containing your protein
over a calibrated gel exclusion column. The emergence of the active
fractions will provide an elution volume that will point you to the
native MW, +/- 20% or so. The difference between a tetramer and an
octamer (2X) should be cake using GEC. (Caveats apply: some proteins
like to stick to GEC columns, but this can be suppressed with 100 mM
NaCl, normally, but YMMV!)
Cheers,
--
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowlett@mail.colgate.edu
Jerry McCully wrote:
type="cite">
Hi, All:
Although it is off-topic, definitely I think I can get some help
here because we crystallographers are dealing with protein purification
almost every day.
My protein was expressed in E.coli as a soluble octamer with or
without his-tag revealed by gel-filtration. For the his-tagged protein,
the final product is an octamer after Ni-column and gel-fil. However,
purification of the non-tagged protein results in a tetramer because of
a partially denatured condition.
When I tested the enzymatic activity , it turns out the tetramer
was active although both of them can be crystallized. Now I am
wonderring whether its native form in human is an octamer or tetramer.
I am planning to purify a little protein from human cells to
verify its native form.
Can anybody recommend some protocols?
Thanks a million,
Jerry McCully
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