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 - that
One piece of advice for speeding up the purification process is to use a
great trick I heard about from Michael Rout, who uses this to trap
protein-protein complexes from yeast:
Pellet your cells as firmly as possible. Go into a cold room, scoop the
pellet into the back of a big syringe (10, 2
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 [m
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:
H
Hi,
Ii happened to me before.
The protein was a dimer and by denaturing the purified protein with
urea, and rerunning the Ni-NTA, I could separate the 2 species. So the
dimer was a mixture of cleaved and uncleaved protein.
If I recall correctly, we then observed degradation again after
refoldi
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 Hoedemaeke
Ive always assumed that "others" in case means the bonds to the hydrogens
added in riding positions during refinement... This also explains that there
are no torsions!
Flip
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Bernhard Rupp
Sent: Sunday, N
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 we
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
Hi Vijay,
It is possible that the difference between the two bands is simply a
methionine ! If you are working with a low resolution mass spec, it
may not be very obvious. In most of the newly synthesised proteins
both in prokaryotes and eukaryotes, the initiator methionine is
removed by
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 transla
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 tri
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 pu
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 rar
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 R
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
signa
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