Dear Phoebe,
We have a constant systems homogenizer that we use routinely as a service
for researchers here at UGA. It is really easy to use and gets up to high
pressure (40k psi) so you can lyse plant cells or other difficult to lyse
cell types. You just pour/pump your resuspended cells, as low
Interesting 'off-topic' thread. I'm a rather long-time user of Microfluidics
cell disruptors (for E. coli or P. pastoris or S. ceresvisiae), and a
shared-use M110-P Plug and Play is used by most of the membrane-heads at my
place. I've generally been happy (or at least not unhappy) with it.
If heat is a concern, you can place your Emulsiflex into a bin with lots of
ice. We have the version with cooling after lysis, but if you are paranoid you
can cool the whole thing.
It has the footprint of a 15” MacBook Pro but it’s way more expensive than that
:-)
Jürgen
We always have used ice with the Microfluidics disruptor. We also, on occasion,
monitor the temperature of the process stream. I'm not convinced, at all, that
there is actually any problem with heating, but I thought it reasonable to put
the query out there. Thanks to all who emailed me.
-MW
Dear all,
Our lab is predominantly interested in membrane proteins and currently
uses a Constant Systems device for E coli hooked up to a cooling unit
which pumps 4C water around the outside of the main chamber and keeps the
sample cool even when using large volumes. This works really well for
Some time ago, there was a nice discussion of cost-effective, wimpy
protein-friendly ways to break open E. coli. We're thinking about replacing an
aging sonicator. If people have a favorite gizmo, could they repeat that
advice?
thank you,
Phoebe Rice
Hi Phoebe -
Cost-effective may not be the applicable word here, but the
Microfluidizer works very well:
http://www.microfluidicscorp.com/index.php?option=com_contentview=articleid=19Itemid=76
This gadget runs on house compressed air (don't try to use a compressed
air tank - you'll empty it
Emulsiflex C5
alternative freeze-thawing, the old way we used to do this spiking with some
Lysozyme
or grinding it in a LN2 cooled mortar, is very effective and pretty cost
effective, assuming you don’t pay for the LN2
Jürgen
..
Jürgen Bosch
Johns Hopkins University
Hi Phoebe,
Another possibility is the Emulsiflex (from Avestin, in canada). Not a
cheap piece of equipment, but very sturdy and efficient to up to 200 mL of
extract (runs on house-air). Can deal with E coli (and even yeast if you
have the models with internal compressor). It comes in 3 sizes I
LOL.
In any case, a French press is also not french, according to Wikipedia it was
invented by Charles Stacy French of the Carnegie Institution of Washington
(hence French press with capital F).
In any case, I agree, Emulsiflex, Constant Systems One-shot, Microfluidizer
etc. is the way to go
Adapted from periplasmic fractionation protocol, but for cytoplasmic
proteins:
1. Incubate resuspended cells with lysozyme in normal buffer but with
20% sucrose.
2. Spin down sphaeroplasts (unbroken cells with no cell wall - ultra
fragile). This also removes periplasmic proteases.
3.
I would also recommend the Emulsiflex line of cell disruptors from Avestin
- it has been my favorite and most easily used instrument of cell
destruction.
On Tue, Feb 4, 2014 at 9:17 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote:
LOL.
In any case, a French press is also not french,
BeadBeater. http://biospec.com/. Gentle, aerosol-free way to break
15-350 mL of cell paste (2-150 g wet packed cells).
Cheers,
___
Roger S. Rowlett
Gordon Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
3 to 1 ratio of bper and yper, 2-3 ml per gram of e. coli plus lysozyme and
bensonaze... I have not used a sonicator in years :) about 1% of proteins
dont like detergents, in which case there are other non mechanical options.
A.
On Feb 4, 2014 10:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote:
board CCP4BB@JISCMAIL.AC.UK (on behalf of Roger Rowlett
rrowl...@colgate.edu)
Subject: Re: [ccp4bb] off-topic: bug busting
To: CCP4BB@JISCMAIL.AC.UK
BeadBeater. http://biospec.com/. Gentle,
aerosol-free way to break 15-350 mL of cell paste
(2-150 g wet packed cells).
Cheers
I'm joining the pig-pile on the Emulsiflex. I like it because:
a. The folks at Avestin are very helpful (they're Canadian--just
naturally nice)
b. It'll break yeast as well as E coli
c. It seems pretty gentle to me (we pack it in ice, so there's
negligible sample
Govindjee and Fork's biography of Stacey French is available here:
http://www.nasonline.org/publications/biographical-memoirs/memoir-pdfs/french-c-stacy.pdf
or if your library subscribes to photosynthesis research,
http://link.springer.com/article/10.1007%2Fs11099-006-0041-6
Mark J van
...@colgate.edu)
Subject: Re: [ccp4bb] off-topic: bug busting
To: CCP4BB@JISCMAIL.AC.UK
BeadBeater. http://biospec.com/. Gentle,
aerosol-free way to break 15-350 mL of cell paste
(2-150 g wet packed cells).
Cheers,
___
Roger S. Rowlett
I will be curious to know about people's experiences with membrane
proteins and lysing yeast cells with the Microfluidizer and how that
compares with using a Retsch Miller, i.e. grinding in a liquid
nitrogen cooled stainless steel chamber and plunging in liquid
nitrogen in between grinding
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