Method for worm protein extraction

Hi all,
Please, can anyone tell me a common method for worm protein extraction?
We are using the following lysis buffer: 10 mM Tris, 150 mM ClNa, 1 mM EDTA, 0,1% NP40 (membrane protein’s detergent), 2 mM inhibitor protease cocktail. pH 7.4.
Do you know an optimized lysis buffer for C. elegans?
How many worms and how you make the shattering of the worms to get about 10 a 20 mg/ml of proteins?

Thanks in advance
Fernando Calahorro

Hi Fernando,

Worms can be sonicated on ice in homogenization buffer [from WormBook Methods chapter: 50 mM HEPES-KOH, pH 7.6; 1 mM EDTA; 140 mM KCl; 0.5% NP-40; 10% glycerol. Add fresh protease inhibitors (aprotinin, pepstatin A, leupeptin, PMSF) and 5 mM DTT before use].

Efficient lysis can also be obtained by vortexing with glass beads or dounce homogenization. Treat with DNase, then centrifuge to pellet the carcasses. A single 10cm plate of worms is more than enough for 10-20 mg of protein.

Reply if you need more details.

Harold

This is the protocol that I’m currently using for Native protein extraction:

  1. Wash a plate of worms (grown on a 10 cm plate) by adding 1 ml M9 to the plate and transferring the liquid to a 1.5 ml eppendorf tube using a glass Pasteur pipette.
  2. Spin down at 6000 rpm for 30 seconds or let the worms settle to the bottom of the eppendorf tube and remove the excess liquid
  3. Add an equal volume of lysis buffer (recipe below) giving a 1:1 ratio between the worm pellet and lysis buffer.
  4. Freeze solution in liquid nitrogen and quickly thaw in a water bath
  5. Sonicate solution on ice

I’ve been using this for small scale extractions that I use immediately for SDS-page/western blots.

Lysis buffer:

50 mM Tris pH 7.5
50 mM NaCl
Protease inhibitor (I use 1 mM PMSF)

-Andrea