Native protein extraction

Lew Jacobson 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 
reproducibly.

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.

Lew Jacobson

> 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
Biological Sciences
Univ. of Pittsburgh
Pittsburgh PA 15260
412-624-4647
Fax 412-624-4759
LJAC at pitt.edu
---




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