Another option is to try NEB NiCo21(DE3) cells.
http://www.neb.com/nebecomm/products/productC2529.asp
I have no relation to NEB beyond being a customer.
They've mutated GlmS to eliminate binding to IMAC resins and have
added chitin affinity tags to SlyD, ArnA and Can to allow simple post-
I
Jw0RyO6IPJmKdwCfbu3S
opLlcpYxP7uzicyX0B6U4aY=
=NH4Q
-END PGP SIGNATURE-
________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
ab / Genes to Cognition
Wellcome Trust Sanger Institute
Hinxton
Cambridge CB10 1SA
Work Tel: 0044 (0)1223 834244 ext. 7318
Cell No.: 0044 (0)7870 208280
===
________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
ould like some suggestions on how to go about
>it. I would also like to try co-expression of GroEL/GroES or
>DnaJ/DnaK, and would like to know where to get the plasmids.
>Any help or comments would be appreciated.
>Weijun
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
NY 14203.
*
________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
nents but seem to be perpetually running out
of the columns.
Thanks
Eric
--
D. Eric Dollins, Ph.D.
C266 LSRC, Research Dr.
Duke University Medical Center
Durham, NC 27710
(919) 681-1668, [EMAIL PROTECTED]
________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
ething similar with DEAE/CM media?
I've
seen references to this (although I suspect the resolution won't be as
good), but not very good descriptions.
thanks,
Nat
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
[EMAIL PROTECTED]
____
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
PROTECTED]
***
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
There is GENtle which has a whole slew of tools associated with it.
There are versions for several platforms.
http://gentle.magnusmanske.de/
If you're used to Vector NTI, it is pretty similar (and open source
for the ambitious).
_
Cynthia Kinsland, Ph. D.
Dir
Using pQE30, any E. coli is an expression host. Because it uses the
T5 promoter, you don't need an E. coli strain carrying the T7 RNA
polymerase (so, you don't need a "DE3" strain).
As noted by Artem, you are most likely having a leaky expression
problem. However, it is odd that DH5a will
I'm not quite sure what you want, but I have a series of vectors
encoding various N-terminal tags and fusions, all followed by a TEV
site. They have an MCS standard to many pET vectors. Therefore, they
are designed to clone your gene in using the NdeI site at the 5' end
(which will, after
2008/C_Structural_Biology.html
______
Cynthia Kinsland, Ph. D.
Protein Production and Characterization Facility
Cornell University
B78 ST Olin Lab Bldg.
Ithaca, NY 14853-2703
Tel: (607) 255-8844
E-mail: cl...@cornell.edu
Incubation with MgCl2/ATP/KCl facilitates the release of the Cpn60.
See: R. E. Joseph; A. H. Andreotti, Protein Expr. Purif. 2008, 60, 194–197.
Best,
Cynthia
__
Cynthia Kinsland, Ph. D.
Director, Protein Production Facility
Cornell University
B78 ST Olin
Since you're seeing the MBP band, it sounds as if you're getting some cleavage.
If there were no cleave you would only see the fusion and TEV bands on the gel.
It may be that your protein is not stable/soluble without the MBP fusion.
Different buffer conditions may help if your protein is bein
ergen"
mailto:jubo...@jhsph.edu>> wrote:
@Cynthia,
On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:
Since you're seeing the MBP band, it sounds as if you're getting some cleavage.
If there were no cleave you would only see the fusion and TEV bands on the gel.
I think that is a wr
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