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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:
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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]
