Previously, I inquired on the C.e. bionet whether anyone had success
with a nitrogen bomb for disruption of C. elegans or had experience
with other useful methods of disruption. I received six emails that
had information on methods and I am now summarizing these responses
for those who are interested. Briefly, Carlos Winter and Eric Moss
recommended breaking frozen worm pellets with a mortar and pestle;
Andy Golden and Gordon Goodall recommended using a bead beater; and
Lew Jacobson recommended the French press. In addition, Carlos and
Lew indicated that the nitrogen bomb does work, but that they now use
other methods. Finally, Marc Perry alerted me to the fact that a
review of biochemical approaches, including the use of sonication, a
cell disruptor, and mortar and pestle to disrupt worms, is presented
in a chapter by Jim McGee and Paul Mains entitled "Biochemistry of C.
elegans" in the book "C. elegans: A practical approach", edited by
Ian Hope.
For our part, we have used sonication, mortar and pestle, French
press, and now the bead beater. The mini bead beater can disrupt
worms quickly (in less than one minute) when we use 0.7 mm zirconia
acid-washed beads. The downside of the approach is that it doesn't
work well with viscous solutions (e.g., 15% glycerol) and it has a
set volume that must be used (1.5-2 ml). We haven't yet tested for
integrity of protein complexes in the disrupted bead beater samples.
For larger quantities, we have been using the French press with some
success for analyzing protein complexes, but in our hands it takes
about 30 min for complete disruption, which is longer than we would
like.
I would like to thank everyone who emailed me and I have included
some of their relevant comments below.
Carlos Winter:
I have used the nitrogen mini-bomb from KONTES to disrupt nematodes,
using the protocol described by Coles et al. (Internat. J.
Parasitol., 19:733-736, 1989) to purify vitellogenin from CEW1 and C.
elegans. It works if you make several passages, but we have finally
decided to freeze the worms in liquid nitrogen and then pulverize the
small pellets formed in a mortar. I don't know if you would be able
to purify any protein by this method, but from the SDS-PAGE profiles
the main worm proteins seem to be solubilized, including myosin. This
protocol can be found at
http://icb.usp.br/~cewinter/rectxt/purifvt_i.html
Just as a curiosity; when I tried to homogenize Panagrellus sp. with
the mini-bomb I found that in the homogenate (after one passage) the
pharynx was intact and separated from the body wall. I have never
observed that in C. elegans or CEW1.
Eric Moss:
We use a cold mortar and pestle to grind frozen pellets of larvae.
It's not really quick, but I feel it is more effective than french
press. We dissolve the powder in a buffer with detergents (DOC and
PTE) to lyse membranes. The nuclei pellet with this buffer, but you
could get extracts of them with modified procedures (I got a nuclear
extract protocol from Rick Roy for run-ons). We dissolve the powder
one-to-one in buffer, so it is pretty concentrated. We know the
extracts are good because we have shown that the ribosomes are
active. We have also used them for co-IPs of mRNP complexes. I think
phosphatases are rampant once the worms are lysed, so you may want to
include an inhibitor, depending on your needs. Our protocol is
published in Seggerson et al. 2002.
Andy Golden:
i have not tried a nitrogen bomb but have been having luck with a
yeast Bead Beater. something very common in yeast labs. instead of
vortexing, this thing shakes tubes with beads and worms and lysis
buffer and does a really good job pulverizing worms, much better than
i have seen with sonication.
we use zirconia/silica beads instead of glass, but you can buy even
more dense beads, like stainless steel.
check out this site for bead beaters, beads, and tubes.
http://www.biospec.com/index.html
we're using 2 ml tubes right now.
Gordon Goodall:
We use a bead beater (biospec.com) to lyse panagrolaimus - not
c.elegans I know but likely to be similar. Good extractions are
achieved in a few seconds but heating can be a problem. It pays to
precool the beads, buffer, sample and apparatus well before use.
Lew Jacobson:
I used a Parr Bomb 20 years ago, with fair success on a moderate
scale. I dimly recall that it is necessary to get up to fairly high
N2 pressures to get very good breakage. There was some difficulty
with the extracts freezing on exit, but this can be avoided. If some
freezing is not a problem, it should work fine. The physical
principle, after all, is identical to that of the French Press. (By
the way, we routinely use the French Press at about 5-6000 psi;
anything below this does not work as well.)
You will need a fairly small pressure cell to do this for co-IP etc.
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Edward T. Kipreos
Department of Cellular Biology
University of Georgia
353 Biological Sciences Bldg.
1000 Cedar Street
Athens, GA 30602-2607
phone: (706) 542-3862
FAX: (706) 542-4271
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