Dear Li,
as pointed out by Marjolein the sample must be MONODISPERSE, rather than
just pure. This means it should be checked not only by SDS-PAGE but at
least by size exclusion chromatography, and be sure that monodispersity
is maintained till the moment of measurements.
I guess your plans are to collect data at synchrotron even if you did
not specify...
In this case you have to check that some critical steps (like freezing
the sample for shipping to the synchrotron) do not affect monodispersity.
As a first check you might want to run a DLS analysis. This should be
performed AFTER sample concentration (if possible up to 10 mg/ml for a
50-100kDa protein) to confirm that this step is not critical. Always
remember to save the exact protein buffer for background data collection
(flow through of the concentration device). An exact background
subtraction is fundamental in order for your further data analysis to
make any sense.
Another step to check is the freeze-thaw procedure because aggregation
might also happen at this stage...
Try flash-freezing your concentrated protein in aliquots, then thaw one
of them to see if DLS confirms that it is still monodisperse. If
everything is ok you can repeat the procedure before measurements,
otherwise you should find a way to avoid these problems. In some cases
you might consider performing the final concentration step (or the
entire protein purification) directly at the synchrotron immediately
before measurements... or to ask for some advice to your local contact.
Ensure also to have enough concentrated protein to perform one
measurement at the highest concentration and 3-4 dilutions at lower
concentrations. After measurements of the concentrated solution you
cannot just recover it and dilute to measure another dataset at
different concentration, essentially because of radiation damage issues.
Hope these advices may help you.
Regards,
Marco
-----------------------------------------------------------
Marco Mazzorana, PhD
Structural Biology and Biocomputing Programme
CNIO - Centro Nacional de Investigaciones Oncológicas
C/ Melchor Fernández Almagro, 3, E-28029 Madrid (España)
Phone: +34 91 2246900 ext. 3033
Fax: +34 91 2246976
www.cnio.es <http://www.cnio.es>
-----------------------------------------------------------
Marjolein Thunnissen ha scritto:
If you go to this web-page:
http://www.embl-hamburg.de/ExternalInfo/Research/Sax/user_info.html there is
ashort summary of the requirements for a SAXS experiment. Shortly the sample needs
to be monodisperse (>90%) and you will need a range of concentrations roughly
from 0.5/1.0mg/ml to 10mg/ml
Marjolein Thunnissen Phone +46-(0)46-22 24584
Associate Professor Fax +46-(0)46-22 24692
Dept of Molecular Biophysics, Lund University http://www.mbfys.lu.se
PO-Box 124 S-221 00 Lund, Sweden
Scientific coordinator I911 (Max-lab): MAD and fixed-wavelength stations
for macromolecular crystallography
________________________________________________________________________
________________________________________
From: CCP4 bulletin board [[email protected]] On Behalf Of Li Sheng
[[email protected]]
Sent: Thursday, April 23, 2009 1:19 PM
To: [email protected]
Subject: [ccp4bb] protein purity in SAXS
Dear all,
Is there anyone with experience in small angle x-ray scattering? Could
anyone please tell me the requirements for protein sample purity and
concentration?
Thanx in advance.
Sincerely,
Li
__________________________________________
Email:[email protected]
Institute of Biochemistry and Cell Biology
Institutes for Biological Sciences
Chinese Academy of Sciences
320 Yue-Yang Road, Shanghai 200031, China
Tel: +86-21-5492-1217
__________________________________________
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