4-year PhD student position in structural biology at the Turku Centre for
Biotechnology, Turku, Finland.
The central focus of the project is to provide insights into the mechanism of
the iron-binding protein Dpr from Streptococcus suis. Understanding the
mechanistic details of Dpr will help in
Hello,
We are trying to purify an N-terminal His6-tagged protein from E.
coli, and the prep was contaminated with the E coli
Glucosamine-fructose-6-phosphate aminotransferase, which co-purifies
with my protein of interest in subsequent ion-exchange and sizing
columns. This protein appears
Hi Ailong,
Use a high salt concentration in your lysis buffer (0.5-1 M NaCl). You
could also try to add some imidazole (e.g. 30 mM). This removes a lot of
unspecific binding to the Ni-column. After applying the lysate I usually
wash with 60 mM imidazole for at least 20 CV and then I elute with
300
Thanks for the suggestions, JJ.
The supernatant was loaded onto Ni-NTA in the presence of 300 mM NaCl
and 5 mM imidazole, washed with 20 mM imidazole (could test higher
concentration...), then eluted with 300 mM imidazole. So I don't
think non-specific binding is a problem. We got 90% purity
If suppression of adventitious protein binding to Ni-NTA with elevated
imidazole concentration is not practical (depending on the protein you
can use up to 50 mM or higher), then you might try separating the
proteins using hydrophobic interaction chromatography, which is rapid
and very
How about trying two opposite ion-exchange columns??!! Namely, monitoring
co-elution behaviour on
anion exchange and cation exchange. For example, we have a case where the
contaminant and protein of
interest will both bind and co-elute from monoQ but only protein of interest or
contaminant
neglecting e-density, packing, etc. - what matters most to people when
running phaser - LLG or Z-score? in what function (RF, TF)?
further, if you 'refine' the RMSD to find the best RMSD, what is the more
robust indicator - LLG or Z-score?
lastly - is it better to have a high LLG/Z-score
In addition to all good recommendations I would add the following: load
your protein to the whatever-Ni column then wash it with urea. 1 to 4 M
urea will not, in most cases, destroy your protein but will disrupt the
hydrophobic interaction between contaminant and your protein in case if
the
Jayashankar wrote:
Dear friends and scientists,
I want to ask about for an integration of modeller in the ccp4i
interface. The interface in ccp4i is designed for modeller Version 6.2,
we have by now modeller 9.2 , which is a lot different.
Nevertheless I tried to enter the necessary data
One other idea idea:
1. Solvent flattening on the hexagonal crystal
2. use the flattening mask to cut out the density of one molecule,
put in a large P1 cell for calculating structure factors
3. Use the structure factors from the density of the hexagonal crystal
to solve the triclinic
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