*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk ***
If your protein gets chopped up in a matter of minutes, try seconds instead - that is not a joke. 30" is a feasable interuption time for proteolysis. On the other hand, maybe your protein contains many loops on the surface. In that case your protein would look chopped down completely on SDS-PAGE even though you still have a stable core. You might try running a gel filtration after short treatment with Trypsin/ Chymotrypsin (those are more specific than Subtilisin) to see whether the core is still intact. This procedure is not uncommon when working with complexes. Make sure you have added enough PMSF to the reaction before loading the column and make sure you thoroughly clean it afterwards - your lab mates will be grateful. -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 13 Jun 2006, Raji Edayathumangalam wrote: > *** 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] > >> > >> > >
