Hi Ed,
yes, this is one of possible ways of doing this. Although I doubt it will
make any (significant) difference in practice compared to other options. All
mentioned methods should normally result in similar values.
Pavel.
On Tue, Apr 12, 2011 at 7:54 PM, Edward A. Berry wrote:
> Pavel Afoni
I thought about HIC too, but do not know if it would work since the
binding of protein to HIC need high salt conc. and I am not sure if the
salt conc. in the sf900 or Hi5 medium is high enough (the formulation is
secret, LOL), thus it is good to know that someone has succesful
experience with
That is exactly what HYDENS is doing. A good interpolation with small
grid steps should be equally good but with current computers and just a
few hundred or even thousand points to evaluate, a classical Fourier
summation is pretty fast and, for me, easier to program than a proper
cubic-spline i
Dear all,
Thanks a lot for sharing, seems that either a HIC column or AS would work, and
that's great, I should give both of them a try.
I thought about HIC too, but do not know if it would work since the binding of
protein to HIC need high salt conc. and I am not sure if the salt conc. in
Pavel Afonine wrote:
Hi Ed,
yes, this is the eight-point interpolation, but since you can select to
choose very small grid step for the map calculation (grid_step
parameter), I hope this should be ok. If necessary, I can add an option
so it will give you the map value at the closest grid point i
So what happened with the non-reducing gel? (If the DTT was fresh,
there should be no problem, but if not...)
JPK
On Tue, Apr 12, 2011 at 3:21 PM, Michael Kenneth Fenwick
wrote:
> Thanks for all your suggestions so far...as a quick reply to some:
>
>>You say the fractions are in equilibrium - ho
Hi Ed,
yes, this is the eight-point interpolation, but since you can select to
choose very small grid step for the map calculation (grid_step parameter), I
hope this should be ok. If necessary, I can add an option so it will give
you the map value at the closest grid point instead of interpolation
Bei,
I had a former labmate who had the same situation and would load somewhere
between 6-8L of media directly onto a column. I don't remember what type of
column it was, ion exchange may not be ideal if the ionic strength of your
medium is high. I think it may have been a phenyl sepharose colu
Without seeing your NZ or L-test plots, but looking at the logs, your
data does not appear to be twinned. It will not be R32 because the
R-merge is too high. The probable space group is C2. The listed twin
fractions will approach 0.5 if your data has perfect twinning or you
processed in too low
Dear all,
My protein of interest was expressed as secreted protein, so I have to collect
the medium and change the buffer with sortorius Jet before I load the sample
onto a IMAC, the buffer change step in my current protocol can last for 12hrs
(I have to concentrate 4L to 200ml, then dilute it
Thanks for all your suggestions so far...as a quick reply to some:
>You say the fractions are in equilibrium - how about keeping the oligomer
>fraction each time and adding it to the subsequent preparation?
I did this once. The equilibrium is sort of a gift that keeps on giving, but
the problem
Do you have a reducing agent in your solutions? I.e., maybe you are
seeing disulfides?
JPK
On Tue, Apr 12, 2011 at 1:27 PM, Michael Kenneth Fenwick
wrote:
> Hi,
>
> I have a protein that shows high and low MW peaks on gel filtration (which
> run at the same MW on SDS-PAGE). There is a slow equi
You might also try to control the degree of oxidation using the microwave, and
setting up trials after different numbers of cycles of heating.
Kendall
On Apr 12, 2011, at 12:41 PM, Jim Pflugrath wrote:
> Frances Jurnak published a paper in 1986 on PEG impurities and purification.
>
>
> As
Hi,
I have a protein that shows high and low MW peaks on gel filtration (which run
at the same MW on SDS-PAGE). There is a slow equilibrium because rerunning the
individual peaks on gel filtration a couple days later shows both peaks. The
higher MW peak is ~2 orders of magnitude more dominant..
Frances Jurnak published a paper in 1986 on PEG impurities and purification.
As I recall, it turns out that different manufacturers put different
additives in PEGs as preservatives. These are generally anti-oxidants.
PEGs do get oxidized.
I suggest you heat up your new PEG solutions to say 80 d
On Fri, 2011-04-08 at 18:06 -0700, Pavel Afonine wrote:
> phenix.map_value_at_point map_coeffs.mtz label="2FOFC" point="1 2 3"
> point="4 5 6"
Cool. Afaiu, this is interpolation. A useful extension would be
automatic picking of (x,y,z) from a pdb-file (a la mapman), although a
determined person
Dear Tim,
On Mon, 2011-04-11 at 10:44 +0200, Tim Gruene wrote:
> since you pointed it out I wonder if there is any reasonable (i.e.
> w.r.t. data
> error/ resolution) difference between the interpolated values and the
> calculated
> value. I actually doubt that
That should depend on the quality o
PS, it might be a good time to start an additive screen.
--
From: "Jun Yong Ha"
Sent: Tuesday, April 12, 2011 7:56 AM
To:
Subject: [ccp4bb] Reproducing crystals.
Hi all,
Recently, I produced crystals with MBClass1-64 which contains PEG4000,
HE
Hi,
Some anecdotes here for your reference:
One paper I read says that the authors were having trouble reproducing a
crystal from an initial screen. After some debugging, they realized that it
was because that they used a same pipette tip when making screens. Adding a
little solution from the
Postdoctoral positions in Bioinformatics and Computational Biology
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or PEG 4000 got old. ask around in the department or university for old PEG
4000 bottles.
good luck!
Berta
On Apr 12, 2011, at 3:28 PM, mickael blaise wrote:
> Maybe your old solution evaporated then you end up in your old tube with a
> more concentrated solution in PEG and NaCl so try to scr
Maybe your old solution evaporated then you end up in your old tube with a
more concentrated solution in PEG and NaCl so try to screen with new
conditions with higher PEG and/or NaCl concentration
Mick
2011/4/12 Jun Yong Ha
> Hi all,
>
> Recently, I produced crystals with MBClass1-64 which
Hi all,
Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na
and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with
different batch of solution. I got the crystals only from 2008 solution, but
not from fresh ones. I asked technical service of Qi
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