I see the issue now, sorry... I agree with Jurgen. Did you get it DNA
sequenced before overexpression? Size of tag?
Jon
On 02/18/2010 03:06 PM, Jürgen Bosch wrote:
Well that it's not his protein which he has crystallized but an over
expressing contaminant :-)
Or the alternative would be although it's supposed to be 29kDa it runs
funny and appears larger in the SDS gel, in that case it would be OK.
If we only had some confirmation that the 43kDa band is the right one
e.g. via Western Blot & Anti-His ...
Jürgen
On Feb 18, 2010, at 4:01 PM, Jon Schuermann wrote:
Maybe I am not getting it, but I don't see your problem or question?
What I understand from your message is that you have a protein that
overexpresses, purifies, and crystallizes... where's the problem? We all
wish we had that problem...
Jon
On 02/18/2010 11:49 AM, Armando Albert de la Cruz wrote:
I am trying to overexpress a His-tagged protein of 29KDa in E.coli
(BL21-codon plus) and I end up with a highly expressed product that of
43KDa that binds to the Ni-column. I also have nice crystals. Does
anyone have any experience on this.
Armando
--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist
NE-CAT, Building 436E
Advanced Photon Source (APS)
Argonne National Laboratory
9700 South Cass Avenue
Argonne, IL 60439
email: schue...@anl.gov <mailto:schue...@anl.gov>
Tel: (630) 252-0682
Fax: (630) 252-0687
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/ <http://web.me.com/bosch_lab/>
--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist
NE-CAT, Building 436E
Advanced Photon Source (APS)
Argonne National Laboratory
9700 South Cass Avenue
Argonne, IL 60439
email: schue...@anl.gov
Tel: (630) 252-0682
Fax: (630) 252-0687
