Hi,
Since you're considering a serious undertaking, it would be good to know
whether your protein is decently expressed and reasonably characterized in
its native state - before advising you on the process :)
If your protein is normally expressed well, is soluble, and not aggregated -
there's goi
Hi All
A little off topic, but I am thinking about expressing and purifying 40+
mutants, for an assay, at maybe 1mg total protein each. I'd like the
purification to be quick and easy (ie. one step) but cleaner than just a
6his-tag purification.
Currently, my options are either GST, a longer
Hi,
I am sorry that I cannot recall the highest concentration of DTT that I ever
used (inadvertently) when working with disulphide-containing proteins.
However I have the following mildly useful suggestion:
If you have properly formed -S-S- as well as a bunch of free -SH perhaps a
safer str
Yong-Fu,
Microsoft in its infinite wisdom decided to bury ActiveX functionality
in Office 2007. To access Controls, you need to click on the big round
button in the upper-left, select PowerPoint Options, choose then
"Popular" tab and then check the "Show Developer tab in the Ribbon"
option. T
Hi,
This is non-CCP4 related question, I apologize to those who are not
interested. But I hope there might be someone over there with a good
answer.
Program: Accelry DS Visualizer 2.0 (Free ware). It has some interesting
features.
My intention: Create an image from pdb files and save as .msv file
I'd agree with Pascal as well, and can add that if you have a protein
for which you use an affinity tag, often high salt (>500mM) will strip
the DNA off your protein, and might even keep it stable.
Cheers -
Miles
On Aug 10, 2009, at 3:59 PM, Pascal Egea wrote:
Hi Neeraj,
An absorption sp
TurboNuclease digests DNA and RNA to 1-4 base long fragments. It's very
useful to remove nucleic acids during protein purification and virus
purification. Another benefit of using TurboNuclease at cell lysis is to
significantly reduce lysate viscosity. So it reduces the lysate volume for
column loa
I had a simple question about DNA binding protein. Is there an
easy way to detect if your heterologously expressed protein is bound to
DNA post purification.
Yes. UV absorbance. DNA absorbs UV strongly, proteins do not. DNA absorbs
260 more thn 280, the opposite is true for proteins. I
I forgot the mention this: does anyone happen to have some pictures of DDM
crystals? It would be very very helpful for me to take a look and get a
sense how they look like. I appreciate your inputs and help.
Thanks,
Joe
On Mon, Aug 10, 2009 at 4:47 PM, Joe wrote:
> Hi all,
>
>
> I am doing
Hi Neeraj,
An absorption spectra between 220 and 400 nm (for example) should show you
if there is DNA coming along with your protein. In theory A280 is about 1.7
times A260 for a pure protein sample. This is a rough estimate. If your peak
is shifted towards 260 instead of 280 then you can suspect t
Two questions RE hardware (dual images on CRT monitor, 3-D with stereo
glasses) stereo on a Macintosh.
1 We have a configuration:
Mac dual Intel processors OS 10.4.11
NVIDIA Quadro FX 4500 graphics card
X11 version 1.1.3
It runs hardware stereo in O and Pymol , which don't require an X-
win
Hi all,
I had a simple question about DNA binding protein. Is there an
easy way to detect if your heterologously expressed protein is bound to
DNA post purification. Also is there an easy way to strip the protein of
DNA without any damage done to the protein in doing so. I would
apprec
Hi all-
Got a perplexing thiol chemistry/phasing issue. To prevent non-native
disulfide bonds from forming between free Cys but to preserve native
disulfides, I have heard of using a very low (say < 0.5 mM) concentration of
DTT. I've recently come across a paper where the assignment of 3 disulfides
Hi all,
I am doing initial crystallization screening for a membrane protein purified
in DDM. The actual concentration of DDM in the protein solution is >> 2 CMC
after the protein concentration step (Amicon MWCO 50 kD used). I just found
over 50 conditions out of 1000 give me crystals. Having w
The paper Acta Cryst. D59 (2003) 2125-2132 discusses searching for
disulfides. In general this works for data truncated for phasing to
between 2.1 and 2.9 A. If the resoltuions is higher than 2.1, you
can search for individual sulfurs, if it is worse than 2.9 even
fitting them as disulfides is un
Hi all
Has there been any work/reports of using disulfide restraints (number
of heavy atoms as well as distance) for heavy atom searching/scoring
for anomalous sulfur phasing?
What resolution range would this be most effective?
Thanks
FR
-
Fra
Hi Donghui,
It’s going to be tricky. Perhaps, you can predict some local rotamer variations
around the ligand interacting regions. Predicting the global changes is
difficult, particularly if you are anticipating a big conformational change.
For example, in one of our structure, the active s
Dear all
i want to know how can i calculate the alpha helical radii of 3-D
structure of a protein. Is there any programme to calculate the radii of
alpha helix.
If you want to display CNS maps in Coot and have them on the right
scale, then you need to do one of two things. Either:
1. Don't use the map file, use the reflection file containing the map
coefficients, or
2. Change the setting in CNS which controls the extent of the output
map. Instead of
Dear Pascal,
2009/8/10 Pascal Egea :
> Dear All,
> I am currently carrying the refinement of a structure and comparing the
> results obtained in Refmac, Phenix and CNS.
> While Phenix and Refmac write maps and their corresponding coefficients in
> mtz format allowing display of 2Fo-Fc and Fo-Fc ma
Dear All,
I am currently carrying the refinement of a structure and comparing the
results obtained in Refmac, Phenix and CNS.
While Phenix and Refmac write maps and their corresponding coefficients in
mtz format allowing display of 2Fo-Fc and Fo-Fc maps in COOT, the
corresponding Fo-Fc map as writ
Hi,
Thanks to the many replies to my queries about cheap/easy alternatives
to Photoshop and CorelDraw on OS X. In a rare display of unanimity,
the bulletin board spoke with essentially one voice:
ALTERNATIVE TO PHOTOSHOP:
Gimp (not gimpshop)
http://www.gimp.org/
As one sage respon
Oops - sorry to mislead you.
Yes - you are absolutely right..
Eleanor
ale...@pasteur.fr wrote:
Just a small comment on Eleanor's instructive advices :
...
If your original structure has cell
(a2= 47.3, b2=58.9, c2=67.6) and angles (alpha2=90, beta2=99.2, gamma2=90)
and the second cell i
Just a small comment on Eleanor's instructive advices :
> ...
> If your original structure has cell
>
> (a2= 47.3, b2=58.9, c2=67.6) and angles (alpha2=90, beta2=99.2, gamma2=90)
>
> and the second cell is
> (a1=67.5, b1=58.8, c1=98.9) and angles (alpha1=90, beta1=101.5, gamma1=90)
> ...
> I woul
First - there doesnt seem much to worry about. The Rs will be higher
than usual when you have a strong pseudo-translation vector. There are
many weak observations for the h k l=2n+1 reflections.
But in cases like this is is very helpful to force the same indexing on
all your different data set
General announcement and how to apply : http://www.farma.ku.dk/index.php?id=6811
Project description: http://www.farma.ku.dk/index.php/Project-10/6840/0/
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Please contact me if you wi
Hi Sajid,
the NADP /NAD binding sites are often "composed" of two sites: one site
that is specific for the adenine/adenosine moiety and another site for
the nicotinamide moiety. It can happen that you see the adenine in the
density but not the nictoninamid tangling around.
HTH Guenter
I ha
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