, October 26, 2012 1:35 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Resuspension of bacterial cell pellets
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
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
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
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.
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
opher.gile...@sgc.ox.ac.ukwrote:
It makes sense to use a fixed ratio of resuspension buffer to cell weight;
we weigh the pellets after centrifugation,
Thanks for the confirmation. Raji
On Thu, Oct 25, 2012 at 10:44 AM, Rob Gillespie
robert.gilles...@duke.eduwrote:
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 opher.gile...@sgc.ox.ac.uk
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
: Thursday, October 25, 2012 5:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Resuspension of bacterial cell pellets
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
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
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
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
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
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
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