Biospec. The chamber caps are dimpled, so when they are tightened, they
displace air and a bit of liquid out the top of the chamber. The amount of
remaining air is very very small if done properly. The chamber should be
completely full of liquid to work as intended.
Roger Rowlett
On Oct 26, 2012 1
Roger Rowlett wrote:
No air in the vessel, no foam.
What manufacturer/model do you use? I can't quite imagine a beater that
would have no air in the chamber but maybe there is something new under the
sun.
Yield of soluble, active protein from broken cells is quite comparable or
better th
No air in the vessel, no foam. Yield of soluble, active protein from
broken cells is quite comparable or better than French press or sonication,
but with no aerosols. The bead-beating unit is encased in ice water, and is
used 15 s on and 45 sec off to minimize heat buildup. The solution still
feel
Roger Rowlett wrote:
This goes straight into a bead beater for complete, gentle homogenization
in 8 min.
Didn't you mean "complete, foam-producing, surface denaturation-inducing"
homogenization? I am not saying that bead beater is worse than the "locally
near boiling temperatures-producing"
We resuspend in a low ionic strength buffer in a 2-3:1 ratio (mL/g). We
typically get 15-25 g of wet packed cells per liter of TB medium, and
resuspend in 40 mL of buffer. This goes straight into a bead beater for
complete, gentle homogenization in 8 min. Protease inhibitors are optional.
We purify
On 10/26/2012 12:29 PM, Francois Berenger wrote:
On 10/26/2012 12:16 PM, James Stroud wrote:
This sounds like something that a PDB file is not intended to do. I
think everyone has universally agreed to the PDB specification at the
RCSB, which makes no provisions for arbitrary objects, as cool as
On 10/26/2012 12:16 PM, James Stroud wrote:
This sounds like something that a PDB file is not intended to do. I think
everyone has universally agreed to the PDB specification at the RCSB, which
makes no provisions for arbitrary objects, as cool as they would be.
I see that there was a proposa
On 10/26/2012 12:16 PM, James Stroud wrote:
This sounds like something that a PDB file is not intended to do. I think
everyone has universally agreed to the PDB specification at the RCSB, which
makes no provisions for arbitrary objects, as cool as they would be.
But you could put your informat
This sounds like something that a PDB file is not intended to do. I think
everyone has universally agreed to the PDB specification at the RCSB, which
makes no provisions for arbitrary objects, as cool as they would be.
But you could put your information into REMARK records, which are free-form.
Hello,
For some new project, I'd like to be able to generate things
and store them in PDB format.
For example, a triangle, a line segment, a square,
a cube, a sphere, an arrow, etc.
Being able to change the color and "line width" would be nice.
Is there some official recommended way of doing th
I typically use a 10:1 ratio of lysis buffer to paste (e.g. 100 ml for 10 g)
for E. coli expression and lyse by high pressure homogenization (e.g. APV at
700-800 bar). I often add Benzonase to the lysate prior to clarification by
sedimentation. This works great for highly expressed proteins with
We are looking for a Research Scientist responsible for our new X-ray
Crystallography Facility at the Institute of Structural Biology of the
Helmholtz Zentrum München. The ideal candidate will have excellent practical
and theoretical knowledge in crystallization and structure determination of
p
Adding TurboNuclease (http://www.accelagen.com/TurboNuclease.htm) to the
lysis buffer can significantly reduce the lysate viscosity especially when
minimum lysis buffer is required. --Chun
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji
Edayathumangalam
Sent: Thursday
We do it differently :-)
1 g cell in 2ml of buffer
Typically we get anywhere between 10-20 g per liter of TB medium. Since we pass
our cells through a cell disruptor and wash afterwards with buffer to maximize
our recovery we end up after cell lysis with about 1g in 3-4 ml buffer roughly
due to
Thanks for the confirmation. Raji
On Thu, Oct 25, 2012 at 10:44 AM, Rob Gillespie
wrote:
> Put me down at another person who re-suspends bacterial cell pellets in
> 4-5 volumes of buffer.
>
>
>
>
> On Thu, Oct 25, 2012 at 9:15 AM, Opher Gileadi > wrote:
>
>> It makes sense to use a fixed ratio
Dear Karolina,
We had one nice example where the protein could easily be concentrated to about
50mg/mL without crystallising, at which point we realised that it would be a
good project for collaborating with our NMR colleagues, i.e. the RING domain of
the Kaposi's sarcoma-associated herpesvirus
Put me down at another person who re-suspends bacterial cell pellets in 4-5
volumes of buffer.
On Thu, Oct 25, 2012 at 9:15 AM, Opher Gileadi
wrote:
> It makes sense to use a fixed ratio of resuspension buffer to cell weight;
> we weigh the pellets after centrifugation, then suspend in at least
It makes sense to use a fixed ratio of resuspension buffer to cell weight; we
weigh the pellets after centrifugation, then suspend in at least 4-5 volumes
(ml/gr) of buffer.
Hi Folks,
Thanks for your responses. To clarify, I have looked into any fluctuations
in cell pellet volumes (autoinduction, cell lysis, toxicity) and this isn't
such a case. My colleague's cell pellet weights are the standard 3g or so/L
and that's why I strongly suspect the resuspension volumes to
In general, I use about 5 mL buffer per gram of pellet, which seems in line
with your usual standards. I would suspect that is a starting point of
your colleagues problems.
In practice, I weigh my bacterial pellet after centrifugation to ensure an
accurate measurement. Bacterial pellet volume can v
On Thu, 2012-10-25 at 11:34 +0100, Eleanor Dodson wrote:
> You can use superpose LSQKAB to fit various residues by number..
> Eleanor
Eleanor is absolutely right.
Coot has Calculate->LSQ superpose option for that.
I feel what needs to be reiterated is that "CCP4 superpose" uses SSM -
secondar
Hello Everyone,
Sorry for this rather naive and non-CCP4 question but I am very curious.
My rule of thumb is to resuspend bacterial cell pellets in about 1-2% of
the original culture volume for a wet weight of about 3g of bacterial
pellet per L of culture volume. For example, Typically, the tota
You can use superpose LSQKAB to fit various residues by number..
Eleanor
On 24 Oct 2012, at 23:59, WENHE ZHONG wrote:
> Dear members,
>
> I have one difficult task on hand and would like to ask for your advice.
>
> I want to superpose two enzyme structures just based on several residues
> (e
On 25 Oct 2012, at 00:45, Zhijie Li wrote:
> This is especially useful when the program does not give you options on
> residue ranges (for example, COOT SSM superpose).
It does but you need to use the function superpose-with-atom-selection from the
scripting interface.
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