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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: > *** 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] >
