Hi all,
thanks a lot for the various responses. When I tried to use a map as the
serach model, I ran into various problems:
again, the starting point is a weak, yet convincing molecular replacement
solution in the hexagonal crystal form (1mol/asu) and no MR solution in P1
(2mol/asu, 2-fold in
Hi,
I have been trying purify a N-ter his-tagged protein over-expressed in
E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS
PAGE which are very close each other (top band in the right MW and more
intense than the lower band). Western blot (for his-tag) of the gel gave
Hi Vijay,
If it is C-terminal degradation, fusing the His-tag to the C-terminus
may help you get rid of it.
Wataru Kagawa
#
Wataru Kagawa, Ph. D.
Research Scientist
Protein Research Group
RIKEN (Physical and Chemical Research Institute)
W221, West
Are you expressing a eukaryotic protein? If so, you might want to
check for rare codons. There are a number of websites where you can
put in your coding sequence and check. I recently had this issue and
it turned out to be incomplete/stalled translation rather than
proteolysis as I had several
Hi Vijay,
It may be caused by the redox status of your proteins, which is normal in
redox related proteins, especially caused by cysteine residues.
The upper band may be the reduced form and the lower oxidized form, which
can be found in numerous papers.
Addition of oxidants or reductants in
The procedure of cutting out electron density, putting it into a large unit
cell, and backtransforming to get structure factors can be tricky (as
you've discovered), so we put some instructions on our webpage:
http://www-structmed.cimr.cam.ac.uk/phaser/density_as_model.html
The last time I
On Nov 4, 2007, at 14:23, Eric Dollins wrote:
Are you expressing a eukaryotic protein? If so, you might want to
check for rare codons. There are a number of websites where you can
put in your coding sequence and check. I recently had this issue and
it turned out to be incomplete/stalled
Dear All -
wondering about what exactly does
REMARK 3 BOND LENGTHS REFINED (A): 2377 ; 0.022 ; 0.021
REMARK 3 BOND LENGTHS OTHERS(A): 2071 ; 0.003 ; 0.020
'others' imply and what does that tell me?
and where can I find the exact meaning of 'period' in
REMARK
Vijay,
It is hard to guess what you mean when you say that 'mass spec results
confirmed both protein bands are the same'. Do you mean that both bands were
cut, digested with e.g. trypsin, and the resulting peptides were mapped? Or,
alternatively, you've done an MS spectrum on the sample that
Others: Atom not included in refinement. If at least one of the atoms
not included in X-ray grad and secder calculation they are marked as
others. These are usually
hydrogens.
Period means if torsion angle has one, two, three or four minimums.
Garib
On 4 Nov 2007, at 20:56, Flip
Some proteases are metal-dependent, and inhibitors for those aren't
Ni-column-compatible. We (meaning my students) found that it helps
to (1) work very quickly and (2) put EDTA into the fraction collector
tubes before eluting from the Ni column.
At 06:22 AM 11/4/2007, Vijay Kumar wrote:
Yes!
And if you have money, you can use phosphoramidon
http://delphi.phys.univ-tours.fr/Prolysis/phosphoramidon.html
It's nearly as good as EDTA or phenantroline for inhibiting metalloproteases
- and it's fully compatible with IMAC!
Artem
-Original Message-
From: CCP4 bulletin board
Another possibility, since you say MS looks identical and you are unable
to separate those two bands by other chromatografic means, is simple a
metal binding site in your protein. If the charge is changed in your
protein due to metal binding then the apparent molecular weight will
differ -
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