I’m really interested in getting nuclear extracts from adult worms, not just embryos. Does anyone here have any experience doing this sort of thing? I found plenty of references to embryonic nuclear extracts but none for adults, so I’m guessing there must be a specific reason why this doesn’t work.
I tried to make transcription extracts from embryos, which has actually been published before and even that didn’t work at all for me. The paper I tried to copy discussed trying to make extracts with mixed stage worms and having no success. I have a feeling that it must just be too difficult to get nuclei out of adults because of all the cuticle proteins, maybe there is another specific reason but in my experience it is probably unlikely to work.
Have you …
J C Vos, H G van Luenen and R H Plasterk G&D ,1993
Preparation of cytoplasmic and nuclear extracts of C. elegans
One volume of worms was mixed with two volumes of nuclear
isolation buffer [NIB: 0.5 M sucrose, 25 mM HEPES (pH 7.5), 25
mM KCI, 0.1 mM EDTA, 0.15 mM spermine, 0.I 5 mM spermidine,
10 mM DTT, 10 mM MgCl2, 0.1 mM PMSF]. This suspension
was ground in liquid nitrogen until a fine white powder
was obtained. Then, four volumes of NIB were added, and the
cells were broken by 40 strokes in a Dounce homogenizer. Debris
was removed by centrifugation at 200g for 2 min. Nuclei
were collected by centrifugation of the supernatant for 5 min in
an Eppendorf centrifuge. The supernatant contained the cytoplasmic
fraction (~ 10 mg/ml of protein). Nuclei were extracted
with two volumes of buffer H containing 500 mM Natl. After
homogenizing in a Dounce, the insoluble material was removed
by centrifugation. The protein concentration of the nuclear extract
was -5 mg/ml.
Gel retardation and DNase I footprinting
Ok, great, I guess I somehow missed that one. Thanks for posting. I’ll try it out.