I should have been more clear. If your protein is insoluble aggregate,
you can use crystal screen results to get an idea of what buffer
conditions favor solubility (and hopefully monodispersity). An example
is described nicely in Collins et al, Acta Cryst F 61:1035.
Ho
Ho Leung Ng
University of
Not sure if it will be helpful... but my protein is not the most
stable protein, in fact, it does aggregate over time (most likely due
to its 'sticky' nature).
However, I still get crystals. The problem is the crystals are among
the gunks and precipitates.
Your case might be different
Dear Raji
Running a blue-native gel with lanes in the presence and absence of a reducing
agent could prove quite informative. DLS could also return a quick result on
the particle distribution in your sample. In that case I would measure samples
as fractionated from the superdex200 and compare
some more thoughts,
Do a cryo-EM imaging, it will be ideal than DLS.
if the particle sizes are uniform i would think your protein in that state
might be useful.
cheers
Padayatti
On Tue, Feb 21, 2012 at 6:21 PM, Raji Edayathumangalam
r...@brandeis.edu wrote:
Hi Folks,
As crazy as it sounds, if
This was meant to Raji,
So here it goes to all.
-- Forwarded message --
From: Zhang, Zhen zhen_zh...@dfci.harvard.edu
Date: Wed, Feb 22, 2012 at 12:15 PM
Subject: RE: [ccp4bb] Aggregated protein for crystallization
To: Pius Padayatti ppadaya...@gmail.com
Hi Pius,
I have done
If you haven't done so already, I would screen buffer conditions
(pH, salt concentration, glycerol, strongly reducing conditions,
ligands, detergents) by DLS to see if you can reduce aggregation. You
might get lucky by setting up crystallization plates, but chances are
you won't get very
You might get lucky by setting up crystallization plates, but chances are
you won't get very useful information from them, especially if your
aggregated protein is soluble.
I seem to fail to understand how crystallization plates would give
information in the not-special case of protein
Hi Folks,
As crazy as it sounds, if you have crystallized and managed to solve the
structure of a protein from aggregated protein, please could you share your
experience.
After many constructs, many many expression schemes and after the usual
rigmarole of optimization that is also often
Hey, it could be that you just have a big oligomer--any support for
that in the relevant literature? A 10-mer would probably beat out an
s200, no? Do you have any other way to ascertain the oligomeric state?
Jacob
On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam
r...@brandeis.edu wrote:
bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Raji
Edayathumangalam r...@brandeis.edu)
Subject: [ccp4bb] Aggregated protein for crystallization
To: CCP4BB@JISCMAIL.AC.UK
Hi Folks,
As crazy as it sounds, if you have crystallized and
managed to solve the structure of a protein from
Hi,
Your idea does sound really crazy but actually Jacob had made a very good
valid point.
Question is do you think your aggregate still functioning or not, if not,
can you revive them in vitro and how effective is your refolding process if
you are going to refold them?
You may want to take a
Well, depends on what 'aggregated' really means. If it implies reasonably
weak oligomerization interaction - and it
might not be too strong given that the oligomers remain soluble - a
chaotropic crystallization agent (on the
extreme end certain high salts, consult Hofmeister for chaotropicity)
Hi,
Did you try using a different column like Superose 6? This column works
well to separate large molecular weight proteins including oligomers.
Ideally if your solution is not cloudy (coming out of void volume) those
are not aggregates those might be oligomers.
HTH,
Shya
On Tue, Feb 21, 2012 at
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