On Wed, 4 Jul 2007 12:36:29 +0200 George M. Sheldrick
[EMAIL PROTECTED] wrote:
The SHELX license agreement has had an 'indemnity clause' in it
for the last 30 years and no-one has complained about it yet! See:
http://shelx.uni-ac.gwdg.de/SHELX/applfrm.htm
I think the reason most folks have
On Wed, 4 Jul 2007 23:21:33 -0700 Tim Fenn [EMAIL PROTECTED] wrote:
I think the reason most folks have problems with the licensing on the
ccp4 *libraries* is that the ccp4 format for maps and reflection files
should be an *open* format - the way it stands now, without writing
your own
Dear all,
Sorry for the off-topic question.
I am purifying a zinc finger transcription factor for crystallization. The
protein appeared as a single band on SDS-PAGE (MW 44KD) after NTA chelating
column, but its OD280/OD260 ratio is as high as 1.0. So I doubt the protein is
nucleic acid
There are several sources.
You can often get them from the manufacturer of your miniprep kit
(the catalog # is written on the side of the bag they come in within
the kit). Many times, they don't list in their catalog that they
will sell it separately, but you can call and get them to give
Hi,
How much salt do you have in your protein buffer?
I would try to increase the salt concetration, during purification.
Hope it helps.
Ana
[EMAIL PROTECTED] wrote:
Dear all,
Sorry for the off-topic question.
I am purifying a zinc finger transcription factor for crystallization.
The
You could also try polyethyleneimine (Polymin P) precipitation to
precipitate out your DNA. Add Polymin P dropwise to a final
concentration of 0.24% to your cell lysate while stirring in the cold
for an hour. This should precipitate out your DNA. Then centrifuge
your lysate and use the relevant
Yes, I have seen colleagues get rid of tons of DNA contamination in bacterial
RNA polymerase preps
by PolyminP (PEI) precipitation. Seemed like it was the only method that worked
(among several
tried) to remove all the non-specific DNA. Worked very well.
As painful as it appeared, PEI
Hi, I think that a good choice, if your protein accept, is to try a gel
filtration at high salt, with or without DNAse incubation before gel
filtration. If your FPLC can do it, a monitoring at 280 and 260 nm will help
you.
Michel
It can also ppt out your protein, if it is bound to the DNA.
You could also DNase and RNase the bejesus out of it, and temporarily
unfold the protein to aid in release of the nucleic acids. Then you need
to get rid of these evil enzymes before you put back your nucleic acid of
choice.
Pavan
We have used the PEI precipitation described by Pavan, very high salt (2M or
even higher) as alluded to by Ana, or heparin columns with success. In some
cases, a denaturing purification protocol can be very useful, but of course
this assumes you can refold the protein. In the case of a
This is off topic but probably of great interest to others.
Where can I find an over-expression plasmid for a high fidelity
thermostable DNA polymerase? I want to make DNA polymerase
to use in a large number of PCR reactions.
Many thanks in advance,
Blaine Mooers
Hi!
With PEI precipitation, the systematic approach will save you time in the end:
do a PEI titration as it is described in the chapter Use of
Polyethyleneminie in Purification of DNA-Binding Proteins by Richard R.
Burgess of Methods in Enzymology, Vol. 208. It takes into account PEI % and
Hi all,
It happened that I had to reinstall my PC laptop; now I need to
get Pymol for WinXP.
I got surprized with the recent changes to get Pymol after Delano
announced the new release 1.0.
I can get the source code but not the executable as free for academic
(if I'm not wrong).
Actually,
Dear Tiancen,
a friend of mine succesfully used a partial denaturation approach using urea to
get rid of bound DNA, see following citation:
Acta Cryst. (1998). D54, 1043-1045[ doi:10.1107/S0907444998000341 ]
Cloning, overproduction, purification and crystallization of the DNA binding
Hi!
With PEI precipitation, the systematic approach will save you time in the end:
do a PEI titration as it is described in the chapter Use of
Polyethyleneminie in Purification of DNA-Binding Proteins by Richard R.
Burgess of Methods in Enzymology, Vol. 208. It takes into account PEI % and
Hi all,
Thanks for all your reply; I got it. I could not reach the link
http://delsci.com/rel/099/ from Pymol Home page in the begining.
thanks,
Ibrahim
--
Ibrahim M. Moustafa,
Ibrahim,
looks like old builds are still available from http://delsci.com/rel/099/ .
HTH,
Kay
Ibrahim M. Moustafa wrote:
Hi all,
It happened that I had to reinstall my PC laptop; now I need to get
Pymol for WinXP.
I got surprized with the recent changes to get Pymol after Delano
Ibrahim,
I got surprized with the recent changes to get Pymol after Delano
announced the new release 1.0.
You can find DeLano Scientific LLC's reasoning at http://pymol.org/funding.html
with a more general discussion at http://delanoscientific.com/about.html
However, the short answer is
Hello,
I am used to purified HIV-1 NC protein which contains two zinc fingers.
Protocol uses PEI precipitation. (See Dynamical behavior of the HIV-1
nucleocapsid protein *Journal of Molecular Biology*
http://www.sciencedirect.com/science/journal/00222836 Volume 279,
Issue 3
I am building a new structure that was partially fit with RESOLVE. Various
lengths of chain are properly numbered (where RESOLVE identified a match)
and the unplaced polyalanine peptides are numbered 801-806, 811-823,
831-835, etc...
When I have identified the correct sequence for a stretch of
Andrew Gulick wrote:
that the PDB file can be re-ordered? I realize I could use a sort command
in UNIX however that would be complicated by the presence of multiple chains
and splitting up the PDB by chain could be as tedious as manually editting.
Any other easy work-around suggestions from
On Thu, Jul 05, 2007 at 02:02:24PM -0400, Andrew Gulick wrote:
I am building a new structure that was partially fit with RESOLVE. Various
lengths of chain are properly numbered (where RESOLVE identified a match)
and the unplaced polyalanine peptides are numbered 801-806, 811-823,
831-835,
You might check how these people did it:
Huang J Lipscomb, Biochemistry, 2006 T-state active site of
aspartate transcarbamylase: crystal structure of the carbamyl
phosphate and L-alanosine ligated enzyme.
PubMedID: 16401065
To me it seems that clause 2.1.1 of the CCP4 academic license says that one
can distribute work derived from or using the CCP4 libraries provided that it
complies with clause 2.1.2
The last sentence in clause 2.1.2 says it itself becomes void if the derived
work is distributed under the GPL or
Dear colleagues:
I added a few lamdas of NaOH and dissolved it. Thank you all for the
responses.
Jackie Vitali
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