Preabsorbing Antisera with C. elegans Acetone Powder

I recommend trying traditional affinity purification first (see my protocol for this) and only using peabsorption if affinity purification doesn't give satisfactory results.

1. Grow a large liquid culture of the desired mutant strain. A good amount is 1 liter of culture, which will yield about 10 ml of packed worms. (see my protocol for worm liquid culture)

2. Starting with a tightly packed pellet of worms, add 3 volumes of 0.1 M NaCl. Lyse the worms by sonication. I've been using the microtip of a Branson sonicator set at the microtip limit. I use 8 minutes of sonication for a 20 ml suspension, with the machine on a 50% 2 second cycle. Examine the sample by placing a drop on a microscope slide and viewing through a dissecting scope. At the end of sonication, about 90% of the worms are lysed, but many worm fragments (and even an occasional live worm) are still present.

3. Transfer the homogenate to a glass graduated cylinder, and add 5 volumes of acetone. Mix by inverting several times; should see a large amount of floculent white precipitate. Allow the precipitate to sediment (takes ~30 min), and remove the supernatant by suction.

4. Wash the precipitate 3 times with 0.1 M NaCl, each time filling the cylinder, letting the precipitate sediment, and removing the supernatant by suction.

5. Fill the cylinder with acetone (to help remove any remaining lipids).

6. Prepare a small (6.5 cm diameter is good) Buchner funnel by placing a circle of Whatmann 3MM paper inside, and wetting the paper.

7. Pour the precipitate/acetone suspension into the funnel and filter it through. Wash through some more acetone.

8. Dry the material overnight at 37° spread on filter paper (actually, a few hours is probably sufficient).

9. Crush the material into a fine powder in a small (5 cm diameter) morter & pestle. Should have a beige powder. Weigh the powder; expect to get about 0.9 g of dry powder starting with 10 mls of packed worms.

10. Pour the powder into a 15 ml tube, and add PBS up to about 15 ml total volume, and vortex to form a slurry. Expect an oatmeal like suspension. Using a broken off pasteur pipette, distribute an amount of slurry that contains the equivalent of 0.1 g dry powder into each of several screw cap 1.5 ml eppendorf tubes. Microfuge for 10 minutes, discard the supernatant, and store the tubes frozen. 0.1 g of powder should be about 300 µl of packed wet material.

11. To preabsorb antisera: mix about 1/3 volume packed moist acetone powder with antibody solution, vortex or mix with a pipette tip to get a slurry, and rotate tube at room temp for 30 min. Spin 10 min in microfuge, transfer sup to a new tube, spin again 10 min, and transfer sup to a new tube. Add sodium azide to 5-10 mM. Store at 4° or frozen for long term storage. Both primary and secondary antibody solutions should recieve this treatment to get the cleanest results.