Hi,
Can I profit from Kristin's question and add one: is it too soon to know if the
crystallography software (CCP4, Coot, Autobuster, XDS) will work fine with
Mavericks (Mac OS X 10.9)?
(I remember a bit of trouble when Lion came off).
Carlos
Em 23 oct. 2013, às 22:10, Kristin Low
Hmmm… is it really (physiological-like) binding, or your protein A is
aggregating/precipitating on the partner? Do you have a good negative control
(a similar protein to which protein A should not bind)? Also, as a general
rule, be careful about your detection method for the pull-down, don't
working...
F
-
Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder
On Oct 23, 2013, at 3:16 PM, Carlos Kikuti kik...@gmail.com wrote:
Hi,
Can I profit from Kristin's question and add one: is it too soon to know
Hi Anita,
You don't state the WL of the melting and back folding CDs, I'm guessing it's
around 210 nm?
In any case, the refolding that you see might be just the formation of
aggregates. For saying if it's really refolding you'd need a new spectrum WL vs
AU in the end of the reverse thermal
Urea?!?!? My arm hair went up.
Anyways, DLS (Dynamic Light Scattering) might help. Hard to imagine something
quicker and easier, provided that someone next to you already has a DLS machine.
(sorry for the late answer)
Carlos
Em 26/06/2012, às 15:10, Brad Bennett escreveu:
Native PAGE
Not sure I understood your problem but it looks like it's related to
flexibility. You can try cutting the domains apart (and chopping off the loops)
in two different pdbs. They can be used as separate ensembles. Try to find the
bigger one first. 35% homology is not that low, just search with
That's good to know, Rhys.
But would you mind sharing why did it work with Balbes? Is there a big change
in the position of the domains as related to your first searching model, or
huge loops that had been removed?
Carlos
Em 02/10/2012, às 14:42, RHYS GRINTER escreveu:
Thanks for your
In think most of the salt bridges I recall in structures are either in the core
of the protein or in interfaces (crystallin or complex interfaces with little
or no polar solvent around). Just like charge interactions, the lower
dielectric constant of the environment makes them stronger.
Hi Abdul
Yes, I had the same bad experience with some of the Fabs I was preparing by
papain digestion several years ago. By the time I just walked around it by
starting with more antibody, yields were less than 20% of the starting antibody
mass (bad as compared to the 60% from the good ones).
I'm replying quite late, so you've probably solved the problem by now, but here
it goes:
I agree with Shane, and I bet your procedure needs LESS aeration (15 ml in a -
I guess - 50 ml Falcon doesn't leave much space for air, and Falcons are
hermetically closed). In BL21 that's usually done for
Hi all,
On the same topic, I'd like to know wether the retina display is also
fine, because I've found complains about X11 (non crystallographic)
programs appearing ugly in it. Is anybody using it?
Just for info, I'm also using Mountain lion without problems ( Fink got
weird but I don't really
4. protein monitoring
Dear Yogesha,
You can use the absorbance at 215 or 220 nm to follow your peptide
during purification. Compounds like DTT and EDTA can increase the
noise at those wavelengths, go to 220 nm if you have things like that
in your purification buffers.
Quantification
Hi, there is a dot plot function in Geneious (www.geneious.com) that
may be useful when the repeats are short.
Carlos Kikuti
Le 5 juin 10 à 01:00, CCP4BB automatic digest system a écrit :
On Fri, Jun 04, 2010 at 08:15:03PM +, Klaus Sengstack wrote:
Dear all,
I am looking
What happens if you load your elution fraction into a size exclusion column? If
your protein of interest comes in the void volume together with most of its
contaminants, you'd better test a different construct, and that's much more
than only changing the tag from N- to C-terminus. Sumo and GST
It's bang on the Y axis I'd just ignore it (treat it as just noise) and
work on the rest of the model. There seems to be some trouble in other parts of
it. (Had a similar case but at 3.5 A data). I wonder what is the gap between
Rfree and Rwork.
And I'm curious for what more experienced
I agree with Mark, except that I wouldn't even try sonication, Triton or
freeze/thaw cycles in that case.
I'd look for emulsification (with a Homogenizer) in a cold room, but if you go
quickly and gently with the French Press (either in a cold room or by using a
cold piston) it might help.
You might be giving too much importance to the THEORETICAL pI of the protein.
If it's supposed to be well charged at pH 7,4 (only a titration curve, and not
simply knowing the pI will tell you this) and it's still precipitating, the
problem might be due to a bad fold, for instance, or to the
the culture volume and move
on.
Carlos Kikuti
Em 26/03/2012, às 14:39, Lorenzo Finci escreveu:
Petros,
It has indeed been speculated that high concentrations of Magnesium and/or
other metals present in the cell lysate effect the binding of the
Histidine-tag, and thus specific conditions
Well GE sells empty columns (XK for low back-pressure, tricorn for higher
back-pressure) that you can fill with the resin you want. They seem
expensive but for that kind of things I don't think any option will be real
'savings'. For nickel affinity we have been having good results with
Roche's
I like the Xbridge columns from Waters, but for SEC-MALS you can also use
Superose 6 Increase (GE), which will take longer to equilibrate and will go
a bit noisier than a silica-based column, but might be advantageous in case
a number of your proteins decide to interact with the silica resin.
Dear CCP4BB,
I am unable to install ARP/WARP on a server with Debian 10, it says:
Segmentation fault
*** ERROR ***
This machine cannot run ARP/wARP executables that
are statically linked to glibc.
*** INSTALLATION OF ARP/wARP 8.0 FAILED ***
Modified version exit status 1
-- after some
://github.com/microsoft/WSL/issues/1462#issuecomment-274398213
>
> Hope this helps!
> Art
>
>
>> On Thu, Sep 3, 2020 at 8:34 AM Carlos Kikuti wrote:
>>
>>> Dear CCP4BB,
>>>
>>> I am unable to install ARP/WARP on a server with Debian 10, it say
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Hello!
I have to admit my maths is a bit lazy, but this discussion got me stitched
up, because of a point I believe has not been addressed: the Rfree flags.
I've been trained to import Rfree flags whenever the crystals have the same
space group and similar cell dimensions to the search model for
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