While staying on the subject, I found the following paper very useful
when trying to produce Se-Met labeled protein in HEK293 Flp-In.
Barton et al. Highly efficient selenomethionine labeling of
recombinant proteins produced in mammalian cells. Protein Sci (2006)
vol. 15 (8) pp. 2008-13
Va
http://www.xtals.org/pdfs/selenium.pdf
(sorry, for some reason first email went out w/o the hyperlink)
Artem
-Original Message-
From: Artem Evdokimov [mailto:[EMAIL PROTECTED]
Sent: Monday, November 03, 2008 9:26 PM
To: 'Jacob Keller'; 'CCP4BB@JISCMAIL.AC.UK'
Subject: RE: [ccp4bb] Semet
By popular request I've placed our modified Se-Met method online:
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob
Keller
Sent: Monday, November 03, 2008 8:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Semet in non-auxotrophic strains (summ
On Nov 3, 2008, at 5:54 PM, <[EMAIL PROTECTED]
> wrote:
If you have received this communication in error
Try this: http://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=ccp4bb&A=1
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Dear 'old' crystallographers,
During one case of structure solution, the data processing programs output
incorrect space group-primitive cubic P4132, which later found out that the
correct one should be face centerd cubic f23. This problem was caused by
perfect twinning.
Now I'd like to invite
Hello all,
Prompted by Kay Diederichs, herein follows a summary of the responses
gleaned from the CCP4BB. The wiki has also been edited to include the
summary:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins
Summary:
1. Protocols for both aux
Dear Friends,
I am sure there are some people who have experience of
using NUCPLOT? I really need to use this program to prepare some figures. But,
I could not download the program from the site:
http://www.biochem.ucl.ac.uk/bsm/nucplot/avail.html. Is there any other way t
Jacob,
We routinely express SeMet proteins in native E. coli without any trouble.
The method is a derivative of Doublie's. If you want, I would be more than
happy to send you a protocol (we've modified the process to gain biomass
yield).
Artem
-Original Message-
From: CCP4 bulletin board
On a less mundane tack, I have noticed that when one of my proteins
crystallized under very banal conditions (10mM NaHEPES 7.0, 1mM DDM, nothing
else), tiny gas bubbles were trapped in the needle shower. I suspect CO2 from
decarboxylation, but can anyone suggest other possibilities, or has anyon
Dear Crystallographers,
I am overwhlemed with all of the responses--thank you all very much for your
help. This listserve is a fantastic resource, and it is great that there is
patience for all of the mundane methods questions! I will try to do my part to
help out when I can.
Thanks,
Jacob Ke
I think we still have better luck with longer, slower, more
gentle soaks - but its crystal-dependent.
Try raising the [PEG] at the same time as you raise the
[glycerol].
Phoebe
Original message
>Date: Fri, 31 Oct 2008 18:57:53 -0400
>From: Artem Evdokimov <[EMAIL PROTECTED]>
>Subjec
You can express Se-Met protein in non-auxotrophs using metabolic
inhibition (Doublie. Methods Enzymol. 276, 523-530, 1997 and Van Duyne
et al. J. Mol. Biol. 229, 105-124, 1993).
The Studier auto-induction paper (Studier, Prot. Exp. Purif. 41,
207-234, 2005) describes an auto-induction Se-Me
Dear Jacob,
You can find several protocols for Se-Met labeling at:
http://www.doe-mbi.ucla.edu/local/protocols
Good Luck,
Art
> Dear Crystallographers,
>
> I have been using an E coli strain called Tuner (DE3) pLacI, which is not
> auxotrophic for methionine, nor is there an auxotrophic analog
Hi Jacob,
you can express SeMet labeled protein in almost any E.coli expression
strain if you use a defined minimal medium.
Here is a receipe that worked quite well in our hands:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins
HTH
Guenter
Dear Cr
Dear Crystallographers,
I have been using an E coli strain called Tuner (DE3) pLacI, which is not
auxotrophic for methionine, nor is there an auxotrophic analogue available.
It may be that my protein can be expressed in other strains, but this one
works well right now, and it is usually better
You are right, that particular video card does not support stereo
graphics. However, with a different card, they should still be able to
make it work with that computer.
http://reald-corporate.com/scientific/graphics_cards_by_os.asp
We were able to pick up an ATI FireGL V7100 for about $130 r
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