*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk ***
Hi, You have to give a few things a go - see what is better. I typically try subtilisin Carlsberg and thermolysin before anything else - these two proteases are sufficiently nonselective. I typically try 5-6 different exposure levels (either by time or by ratio) per protease, then SDS-PAGE and MS the results. Here's an example of what has worked (with a gel picture, too!) http://www.xtals.org/pdfs/FliC_FliS.pdf Artem ex-PGP Original Message: ----------------- From: Pirkko Heikinheimo [EMAIL PROTECTED] Date: Tue, 13 Jun 2006 10:51:42 +0300 To: [EMAIL PROTECTED], [EMAIL PROTECTED], [email protected] Subject: Re: [ccp4bb]: boundaries of new constructs *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** 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: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > 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: > >> *** For details on how to be removed from this list visit the *** >> *** CCP4 home page http://www.ccp4.ac.uk *** >> >> >> 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] >> -- &&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& 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 &&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& -------------------------------------------------------------------- mail2web - Check your email from the web at http://mail2web.com/ .
