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---------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----------
