Re: AW: [ccp4bb]: boundaries of new constructs

[james whisstock]

V8 at 4C or room temperature is also sometimes a goer - trypsin is a bit of a horror since it can really chew things up. J [EMAIL PROTECTED] wrote: *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** We routinely try Trypsin, Chymotrypsin, papain and elastase 1st trial is usually 4°C incubation and taking aliquts at several timepoints (30 min - overnight). when we see cleavage in SDS PAGE we continue as Tim lined out identification of stable fragments has worked in several cases (including trypsin:-) best wishes alex Dr. Alexander Pautsch Protein Crystallography /Structural Research Boehringer Ingelheim Pharma GmbH & Co. KG Deutschland Birkendorferstrasse 65 88400 BIBERACH, Germany tel. +49 - (0)7351 - 54 4683 fax. +49 - (0)7351 - 83 4683 email [EMAIL PROTECTED] -----Ursprüngliche Nachricht----- Von: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED]] Gesendet: Dienstag, 13. Juni 2006 09:52 An: Tim Gruene Cc: Wang, Yeming (NIH/NIEHS) [F]; ccp4bb@dl.ac.uk Betreff: 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]