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It would be nice if those who have had success on limited proteolysis
could share which proteases they are using. We've tryed limited trypsin
digestion but mostly end up with total hydrolysis or no visible effect
(the target proteins have natural protease sensitive sites which we
expect to be proteolysed and thus the effect be visible on a gel).
with best regards,
Pirkko Heikinheimo
Tim Gruene wrote:
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I would recommend restricted proteolysis, i.e., a time series of digestion
with various proteases. Start with concentration ratios of 1:500
protease:your_protein and check the success by SDS-PAGE. The N-terminus
can be determined by N-terminal sequencing. For the C-terminus you can try
to scale up, run a gel filtration column or (probably better) an ion
exchange column + GF and do a mass spec.
If mass spec does not work (proteins soluble at high salt concentrations
only are a hinderence or if the domains are not well defined by the
proteolysis), you can estimate the weight from SDS-PAGE and make use of
the secondary structure prediction, available e.g. through the
expasyserver (www.expasy.ch).
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Mon, 12 Jun 2006, Wang, Yeming (NIH/NIEHS) [F] wrote:
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Sorry for the off-topic question!
My current construct of a new protein (has some homologs) doesn't seem to be good
and I have to make some new ones. I am just wondering how to determine the
N-terminus & C-terminus of new constructs if I am only interested in structural
study of a few domains. Any good methods or softwares to recommend? Thanks a lot!
Yeming
---------------------
Yeming Wang, Ph.D.
Laboratory of Structural Biology: Macromolecular Structure Group
National Institute of Environmental Health Sciences
National Institute of Health
Mailing Address: Street Address:
NIEHS, MD F3-05 NIEHS, Building 101, Room F362
P.O. BOX 12233 111 T.W. Alexander Drive
RTP, NC 27709 RTP, NC 27709
Tel (o): 919-316-4634
E-mail: [EMAIL PROTECTED]
--
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Pirkko Heikinheimo
Structural Biology and Biophysics,
Institute of Biotechnology,
P. O. Box 65,
FIN-00014 University of Helsinki, Finland
Visit address:
Biocenter 3, room 4320
Viikinkaari 3, 00790 Helsinki, Finland
http://www.biocenter.helsinki.fi/bi/xray/pirkko
e-mail: [EMAIL PROTECTED]
phone: 358-(0)9-191 58957
gsm: 358-(0)50-354 0713
fax: 358-(0)9-191 59940
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