Dear Amit,
You are working with a membrane protein and the use of SDS (harsh detergent)
often makes these proteins to oligomerise. Boiling your sample is not
advisable, it might make it worse.
I’m not sure why you would like to “see” a monomer in the gel but if you really
would like to know
Dear Amit,
Maybe try adding an equal volume of 8M urea to your sample before
adding the SDS-PAGE sample buffer. Then I'd test boiling and not boiling
that sample prior to loading on the gel.
Good luck,
Mark
On 21 February 2017 at 17:22, amit gaur wrote:
> Hi all,
> I am trying to pu
Ethan and Phil,
Your responses about i) normalization based on minimizing the average
differences between Fobs and Fcalc and ii) the smearing of electron density
by various physical phenomena, makes sense.
The smearing cannot easily be accounted for and having density, where it
should not be, can
On Tuesday, 21 February, 2017 16:53:06 Hunter Moseley wrote:
> Is there a straight-forward way to estimate the amount of missing electron
> density that a particular protein structure is missing based on the
> difference between Fo and Fc?
Short answer: no.
> It appears that the normalization of
Why do you need to “do” anything? Is there some reason you would like to see a
monomer on your gels? It is common for membrane proteins to run as non-covalent
oligomers in SDS-PAGE, and sometimes boiling makes it even worse. I think KCSA
runs as a tetramer on gels, and several proteins I have wo
Hi all,
I am trying to purify a potassium ion channel from insect cell using
baculovirus expression system. I am not seeing monomer of this protein in
SDS instead a dimer appears.So,I increased DTT in SDS buffer but no change
and dimer was intact. In size exclusion this protein appeared as a
Is there a straight-forward way to estimate the amount of missing electron
density that a particular protein structure is missing based on the
difference between Fo and Fc?
It appears that the normalization of the Fc due to the employing of a
maximum entropy method that keeps Fo and Fc comparable
Dear Paul,
As you suggest, small molecule crystallographers are used to such
complicated situations and often use SHELXL for the purpose. Since you
fortunately have 1.0A data you could do the same, first using pdb2ins to
convert your PDB file to a SHELXL .ins file. You will need to use PART
i
We've been working on a high resolution (1.0 A) DNA structure that has
several coordinated magnesium ions that have complete octahedral
geometry via water or phosphate oxygens but are clearly in multiple
conformations (which is related to the multiple conformations of the
coordinating phosphate
Ps - application deadline is the 13th of March, 2017.
Best,
-Joseph.
Joseph Brock | PhD
Division of Physiological Chemistry II
Department of Medical Biochemistry and Biophysics
Karolinska Institutet
Scheeles väg 2
SE-171 77 Stockholm, Sweden
From: Joseph Brock
S
Dear colleagues,
Please see the information below regarding an opening in our research group.
Please share with anyone who might be interested.
Thanks!
-Joseph.
A two-year post-doctoral position is available at the Department of Medical
Biochemistry and Biophysics (MBB),
Could you rename it for the time being - ASN or LEU for ASP or some such
cheat?
Not sure whether local NCS requires identical residue names as well as
conformations..
Eleanor
On 21 February 2017 at 10:39, Alice Dawson (Staff)
wrote:
> Dear all
>
> I am working on a structure with 10 monomers in
Thanks
Kay and Graeme for inputs.
I should, nevertheless, go deeper into this. I should say that,
in my experience, in some cases the effect is small, but in some
others, I would consider significative. I will try to be more
systematic when possibl
Dear Kay,
Thanks for your input.
My intention is to compare a number of datasets, to what
resolution they extend to, based on a common criterion (which
might be I>sig(I) or even CC1/2), but I think to make the
comparison more "equa
Dear all,
eBIC and CCP-EM are proud to announce the inaugural symposium of eBIC, the
UK national cryoEM centre at Diamond Light Source, and the third CCP-EM
Spring Symposium on April 24–26, 2017. This three day conference will be
held at the Diamond/RAL Campus, Harwell, Oxfordshire.
The eBIC open
Dear all
I am working on a structure with 10 monomers in the asymmetric unit (2
pentamers). I am using the NCSR local option in Refmac to automatically
generate NCS restraints. This is working well (much better than the NCS
restraints I defined manually). However there is one residue in one sub
Dear Eike,
What is the interest of soaking experiments when you can grow HEWL crystals
in less time (15 min) than it would take to do a soaking experiment?
https://www.hamptonresearch.com/product_detail.aspx?cid=28&sid=173&pid=524
Soaking will work as long as you do it correctly. A 20min soak or
Dear Jorge
As a rule of thumb I would always integrate every reflection on the detector
face & only limit the resolution in scaling (*1) - most integration programs
are well behaved when it comes to modelling reflection profiles & integrating
spots which are “invisible”
As to why you observe w
I've also experienced this, but since the improvement is small, I did not pay
much attention, and did not investigate. My hypothesis why this occurs agrees
with yours. Nothing should prevent you to make use of this effect!
best,
Kay
On Mon, 20 Feb 2017 08:24:58 -0300, Jorge Iulek wrote:
>
>
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