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Preabsorbing Antisera with C. elegans Acetone Powder
by Michael Koelle, adapted from "Antibodies: A Practical Approach" by D. Catty
One way to eliminate crossreactivity of a polyclonal antiserum raised against a worm protein is to preabsorb the antiserum against total protein extracted from a worm mutant that does not produce that specific protein. Antibodies which bind "nonspecifically" to other worm proteins might thus be eliminated, while antibodies that react only with protein of interest will remain. This method, in addition to its simplicity, has a theoretical advantage over traditional affinity purification of antibodies. Affinity purification involves binding antibodies to the specific antigen bound on a solid support, washing, and then eluting the antibodies with partially denaturing conditions. This elution can fail to elute the highest affinity (and therefore most desirable) antibodies, and can also permanently inactivate some sensitive antibodies. Cleaning up an antiserum by preabsorption, on the other hand, does not suffer from these problems.
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.
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果蝇头部收集--DROSOPHILA