I tried the mortar procedure, and found it took forever, with little
chips of thawing yeast inevitably hopping out and sticking wherever,
since everything was so brittle, even though I had extruded the
pre-frozen pellet through a syringe into liq N2 to make
yeast-pellet-noodles first. In contrast,
I am not aware of this point being explicitly made. Maybe somebody else
could point to the relevant reference?
However, the logic here is very simple:
(1) Take a model and generate from it Fc
(2) Calculate map with minimal rms error m*Fo*exp(i*phiCalc)
(3) This map is biased with respect to err
Hi Cory,
I am afraid to say that Pichia and Saccharomyces are tough cells to break
compared to bacteria. We also purifiy membrane proteins in yeast in my lab
so we have gone through this process.
With regular yeast we can break in an emulsiflex but it takes some hard work
and this is not really via
Yes that's how it's conventionally done if you are working with plants.
Instead of a drill (what did safety inspection said to that, did you have
proper precautions in place etc.) they use a mortar cooled in LN2 and also
containing LN2 within the mortar and a suitable grinder plus cryo gloves (fo
I know this seems a bit crazy, but I used to lyse s. cerevisiae by
pelleting the cells into a 50mL conical, freezing in liquid nitrogen,
and using a power drill with a drill bit fitting the caliber of the
tube, all at liquid nitrogen temp through periodic (or constant)
re-immersion in nitrogen. It
You shouldn't have clogging up problems in the first place. We don't use Pichia
but have the same system and I've used it before at the MPI in Martinsried. The
system is designed that there are no parts which break, you only have to
maintain it on a regular basis and clean it properly.
Do you ha
Hello all;
I have successfully expressed a membrane protein in Pichia pastoris,
however I am having a difficult time with cell lysis.
I have used a Avestin emulsiflex to lyse them, however I have had many
difficulties with the system clogging up, and parts wearing out with the
high pressures.
So
Thanks all of you for your answers. As you guessed I was doing coomassie
staining from single crystal (0.10-0.13-0.05).Fortunately I had lots of not
so great looking single crystals from similar drops and I took about 10-12
of them.Then I could see a faint band where I was expecting!
Thanks aga
I am using the glutathione sepharose 4B beads from GE. I had noticed that the
capture efficiency does decrease with usage, though I have successfully
regenerated the beads at least 20 times and I am still using them.
I regenerate using 2CV of 6M guanidine after each run and every 5 runs with 70
Thanks! Can you refer me some documents about your following statements:
derivation of sigmaa-weighted 2mFo-DFc formula is by calculating Fourier
coefficients of the following map:
Rescaled composite omit map, where minimal structural element (of the size
about the resolution element) is being omi
Hi,
There is no way you can compare NiNTA and GSH affinity supports.
It is a lot easier to regenerate NiNTA.
This has to do with the ligand, not the support.
Kd is 10-100 times lower for GSH.
GSH contains a "highly" reactive SH group if enough thiolate is present
(oxydation and/or nucleophilic att
I have read the paper by Gill in Acta Crys F66: 364-372 and wonder
what other peoples experience has been with the MUVIS from
Formulatrix and the JANsci microscope for imaging 96 well plates.
The JANsci instrument seems to have an advantage over the MUVIS in
having two objectives. For detec
Try RasMol 2.7.5, e.g.
load ../data/pdb1w0k.ent
restrict not hoh
map generate LRsurf dots
map select atom within 1.8
show selected
=
Herbert J. Bernstein, Professor of Computer Science
Dowling College, Kramer Science Center, KSC 121
Hi,
You first need a program to calculate ASA, e.g. Naccess.
Once you have the ASA on a per-residue basis, you can plot a histogram, by e.g.
feeding the data to a spreadsheet application of your choice.
What is usually done is to list residues sequencewise vs. ASA.
Best success,
Nadir
--
Pr. N
Hi,
Well you did not mention which company makes your GST sepharose, but I
used GE GST Sepharose 4B quite often and was able to reuse it quite a
few times.
Chris
On Tue, 2010-11-02 at 11:46 -0400, Mirek Cygler wrote:
> Hello,
> For various reasons we are frequently expressing proteins wit
Hi Ivan,
there are several tests (e.g. Izit dye, crush test) you can do discern
protein from salt crystals but what was always very informative to me
(and certainly in the case of complexes) is a silver-stained SDS-PAGE
gel of the crystals using the following protocol:
- select a drop whi
I recently ran mass spec analysis on some crystals that I had obtained
from an optimization screen. I was looking for modifications in the
protein. In order to get enough signal, I had to harvest and dissolve
about 8 crystals roughly 0.3 x 0.15 x 0.15mm into the MS loading buffer
in order to get a
A2220
Sigma
ANTI-FLAG® M2 Affinity Gel
On Nov 2, 2010, at 11:01 AM, Oganesyan, Vaheh wrote:
> On the same note with Mirek: does anyone know of a source other than GE for
> resin for purification of FLAG-ed proteins?
>
> Thanks.
>
> Vaheh
>
> -Original Message-
> From: CCP4
On the same note with Mirek: does anyone know of a source other than GE for
resin for purification of FLAG-ed proteins?
Thanks.
Vaheh
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mirek
Cygler
Sent: Tuesday, November 02, 2010 11:47 AM
T
Hello,
For various reasons we are frequently expressing proteins with a GST
tag. The glutathione sepharose beads that we are using for affinity
purification seem to be difficult to regenerate and we see much lower
capacity when used the second time. We are following the manufacturer's
instr
Hi,
As far as I know, thanks to Ian, areaimol in its latest versions now has an
option of showing the fraction of accessible area per residue (relative to its
accessible surface in the Gly-X-Gly peptide) both in the log file and in the
B-factor column of the pdb file it prodcues.
Cheers,
Hello Buz,
I do not know what you mean by 'linear map', but according to its manual, the
ccp4-program "surface" writes a list of accessible are per atom per residue,
which you could convert into the total fraction per residue with not too much
effort.
Is this what you are looking for?
Cheers, Ti
Dear All,
I'm looking for a software program to produce, given a 3D atomic structure of a
molecule, a linear map showing the surface accessibility of residues in a
protein structure.
Would any one know of a program that can produce this sort of map.
Thanks! and all the best,
--Buz
The other thing you can try to do is "the-cheap-men-silver-stain"
scan your gel and bump up the contrast, you'd be surprised what you can detect
even from a regular stained gel.
Best results are if you make the gel first as a greyscale image.
Google for Neuhoff stain and bump up the phosphoric a
It reads like you need to run a lane or two with a positive control of some
kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other
crystals of a protein around the same expected molecular weight and try run
on the gel lanes with about the same amount of crystalline volume as your
puta
Dear All
New version of jligand - version 1.0.0 is now available to download and use. It
can be downloaded from
http://www.ysbl.york.ac.uk/mxstat/
There are also tutorials how to jligand to create ligand and link descriptions.
jligand is a program to create new ligand descriptions. It also ca
I'll add it at some stage
Phil
On 2 Nov 2010, at 09:38, Ian Tickle wrote:
> I would say that being able to take in phases & H-L coeffs would be a
> useful option, because sometimes (I accept it's not mandatory) phases
> are available in SF files from PDB which allows us to see exactly the
> same
I would say that being able to take in phases & H-L coeffs would be a
useful option, because sometimes (I accept it's not mandatory) phases
are available in SF files from PDB which allows us to see exactly the
same map that was interpreted by the depositor. In some cases there's
some doubt that th
As Ian says, it doesn't modify the reference set.
Yes it is a subsidiary function of Pointless, aimed at replacing the program
REINDEX (since much of the functionality was already in there, it seemed
sensible to add this option). It ought to do appropriate phase changes, but
like Reindex, it do
Re-indexing of an input (either merged or unmerged) dataset to a (also
merged or unmerged) reference dataset is one of the 'subsidiary'
functions of pointless, according to Phil's man page:
This mode is selected if an HKLREF dataset is specified.
Given a test dataset, merged or unmerged (fil
CAD and Kevins phasematch correctly change phases etc when you change
symmetry operator.
I cant think that this is the job for pointless.. it is responsible
for intensities, and surely only needs to use a merged file to decide on
the appropriate choice of axes - eg getting your new PG3 data
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