线虫蛋白提取--Making Protein Gel Samples from Worms

Making Protein Gel Samples from Worms

by Michael Koelle

1. Grow up 3-5 large plates of worms. This should give ~200 µl of packed worms after the washes described below.

2. When the worms have almost or have just starved, wash the worms off the plate by dumping a little M9 medium on the plate, swirling gently, and sucking the liquid off with a Pasteur pipet. Spin in a clinical centrifuge to pellet the worms, and discard the sup.

3. Wash the worms once with M9, spin down and discard the sup.

4. Transfer the worms in a small amount of liquid to an eppendorf tube, spin briefly at 3,000 rpm, and remove as much liquid as possible.

5. At this point can freeze the worms at -80° if you want to wait for other strains to catch up.

6A. If you don't need embryos in your sample, can just add 1 ml gel sample buffer and boil the tube (use a screw cap eppendorf) for 5 minutes, spin the tube 10 min. to pellet debris, and transfer the supernatant to a new tube. Boiling will dissolve the adults, but the eggs will not be lysed.

6B. If you want to have embryos in your sample, put the worms in a 2 ml eppendorf tube. Add protein sample buffer to the worm suspension until there is a total of 1 ml in the tube (the tubes have a 1 ml mark on the sides). Using a probe sonicator with a microtip, sonicate for 20 seconds. Using our Branson sonifier, I set the machine at the microtip limit, on a 2 second 50% cycle. Make sure the tube is in a beaker of icewater during sonication to keep it from heating up too much. If the sonication is done correctly there should be absolutely no frothing. Examining the tube in the dissecting scope, you should see almost no worms or debris left. Spin the tube 10 min. to pellet debris. Transfer the sup to a 1 ml screw cap tube.

Note: Mark Metzstein thinks it may be possible to lyse eggs by freeze/thawing 3 times in a sample buffer lacking glycerol, which might be easier than sonicating. I haven't tried this.

7. Store the sample at -80° - these samples seem to get degraded when stored at 4° for a few months.

8. About 30 µl of sample should be loaded on a 1.5 mm thick gel using 5 mm wide sample wells. This should be about the maximum amount of protein that will run well without streaking. If you need to assure even loading of several samples you should load dilutions of your samples on a gel and do a Coomassie stain to determine what volumes of your various samples contain equivalent amounts of protein.

Sample buffer: 100 mM Tris, pH 6.8, 2% SDS, 5% ß-mercaptoethanol, 15% glycerol, enough bromophenol blue to make it dark looking, and optional: 4 M urea. Store in a tightly sealed container to keep the BME from going off. You may still need to add more BME if your solution is old, since it is highly volatile. Some people keep sample buffer without BME and add it right before use.

上一篇：线虫基因组杂交-Worm genomic Southern blots   下一篇：Generating males by heat shock(worms)