I want to generate a ‘crude’ cellular extract, including membranes, from N2 worms, HPLC assay a specific enzyme activity and then compare with deletion strains lacking genes encoding one or more enzyme homo/paralogues.
Is physical homogenization possible in a Potter style grinder or do I need something stronger - probe sonication, bead blasting?
We have used a yeast bead beater (Biospec Mini-Bead-Beater) with good results.
Beat beader worm lysis protocol
Lysis buffer:
TrisHCl pH 7.4 25mM
Sodium chloride 125mM
TritonX-100 1% (or other detergents of your choice) (Omit detergent for membrane fraction analysis experiments. For membrane analysis, do more rounds of bead beating, depending upon the stability of your protein of interest. Worms have been lysing less efficiently in the absence of detergent)
➢ Worms are thoroughly washed with cold M9 and spun down at about 1800g for 2 minutes. Perform the wash 3X times (2X with cold M9 and 1X with ice cold lysis buffer).
➢ Give one final wash with cold lysis buffer with protease inhibitors (we use 1 tablet of Roche complete mini-EDTA free protease inhibitor cocktail with 10 ml lysis buffer).
➢ Final worm slurry should have worms +equal volume of lysis buffer.
➢ Check the concentration of well mixed slurry by putting about 10 ul on a cover slip under a dissecting microscope. This gives an approximate idea of the density of worms in the slurry.
➢ A 2ml polypropylene tube (with O-rings in the cap) is filled halfway with beads. We have been using the 0.5 mm Silicon Zirconia beads.
➢ The bead beater should be pre-cooled in a cold room.
➢ We have not tried more than 1 ml slurry in one tube. (500 ul packed worms + 500 ul lysis buffer). Ensure that the tube is filled to the rim with buffer. This is in order to protect proteins from the shearing force of air bubbles.
➢ Run the bead beater for 20 seconds.
➢ Put the tube in an ice water bath for 2 minutes.
➢ Repeat the bead beating followed by cooling until you observe ~80% reduction in intact adult worms (tested with 10 ul slurry on a slide).
➢ When using detergent e.g. 1% TritonX-100, about 5 bead beat cycles are sufficient for lysis.
➢ Measure the concentration of the worm lysate by reading absorbance at A280. An approximately 2 mg/ml protein concentration was sufficient for our experiments.
Some other methods for lysis are summarized at the following link:
http://www.bio.net/hypermail/celegans/2003-April/002622.html
does this buffer lyse anything, also bacteria that are coming with the worms? or does it only depend on the size of the beads?
The beads come in different densities for lysing different materials. I don’t know if the E. coli are lysed in this protocol, but I would be surprised if they were not.
If you include detergent, the bacteria will definitely be lysed (independent of bead size).