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Sorry about the blank e-mail earlier ! Just in case it helps and you havent seen it yet, there is an article in one of the volumes on macromolecular crystallography in the Methods in Enzymology series. Koth CM, Orlicky SM, Larson SM, Edwards AM. Use of limited proteolysis to identify protein domains suitable for structural analysis. Methods Enzymol. 2003;368:77-84. Best, Anirban. On Tue, 13 Jun 2006, Anirban Banerjee wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > > > ---------Included Message---------- > >Date: 13-Jun-2006 11:47:07 -0400 > >From: "Raji Edayathumangalam" <[EMAIL PROTECTED]> > >Cc: "Wang, Yeming (NIH/NIEHS) [F]" <[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 *** > > > > > >My protein gets chewed to pulp in a matter of minutes in even > 1:4000 > >ratio of trypsin:protein. > > > >I've had to play with incubation temperature, time and > >enzyme-to-substrate ratios to capture the protelytic fragments. > > > >And, like others suggest - N-terminal sequencing, mass spec, > compiling > >info from secondary structure prediction and sequence alignments > of the > >protein from multiple species was my way to enter the > hit-and-miss world > >of cloning a gazillion constructs thereafter. > > > >HTH. > >Raji > > > >-- > >Raji Edayathumangalam > >Postdoctoral Associate > >The Rockefeller University > >Box 224. 1230 York Avenue > >New York, NY 10021 > > > > > > > >> > >> > >> 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] > >>> > >>> > > > > > > > > > ---------End of Included Message---------- > >
