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Yep. I had to cut down drastically on time and enzyme amounts as well.
That worked.
Yes, based on all that we know and all that we gathered, my protein is
expected to be multi-domain with loops etc.
My digests were diagnostics for cloning. Will know soon :-)
Raji
Tim Gruene wrote:
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:
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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]
--
Raji Edayathumangalam
Postdoctoral Associate
The Rockefeller University
Box 224. 1230 York Avenue
New York, NY 10021
