Native protein extraction
ljac+ at pitt.edu
Mon Apr 19 03:28:05 EST 2004
Like Dr. Lye, we use and like the French Press, but only for fairly
large-scale extracts (10-50 mL). There is also a device called a Parr
Bomb that also works by decompression like a gas-driven French Press.
Sonication is more practical for smaller volumes, but very hard to do
For much smaller amounts (e.g., 10-50 animals) we pick counted worms
into phosphate buffer containing 0.1% Triton X-100, then do 6 cycles of
fast-freezing (liquid N2) and slow thawing in ice-water. Finally we put
the extracts on a platform-type vortex mixer for 1-2 min. to finish
breaking the carcasses. The vortex step greatly improves the
reproducibility from sample to sample. See Zdinak et al., J Cell
Biochem 67:143-153 (1997).
I fully agree that proteinase inhibitors are a Good Thing. Most
important ones are leupeptin, pepstatin and MG132.
> Can anyone point our group to a protocol to extract NATIVE protein
> (i.e. no Tri-Reagent/Trizol phenol-solubilized protein) extraction from
> C. elegans? We obviously wish to avoid using proteinase K to lyse the
> worms. My colleague wishes to perform kinetic assays on an enzyme from
> worms. Thanks very much in advance for any help.
> Oscar Aurelio, Ph.D.
> Assistant Professor
> CSU Fullerton
> Dept. of Biological Sciences-MH282
> 800 N. State College Blvd.
> Fullerton, CA 92834-6850
Dr. Lewis Jacobson
Univ. of Pittsburgh
Pittsburgh PA 15260
LJAC at pitt.edu
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