C. elegans Immunoprecipitation¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
Immunoprecipitation protocol for use with C. elegans worm lysate
- Preparation of worm lysate:
- Add 2 ml of PLC buffer to 0.25 ml of worm pellet.
- Break up the worms by sonicating.
- Spin 10 minutes @ 13000 g in microfuge, save supernatant.
- Quantitate protein in lysate using Pierce BCA kit.
- Wash 100 ml (actually 10 ml of beads since it comes as a 10% suspension) of Protein A Sepharose in PLC buffer. Spin 3 minutes @ 13000 g in microfuge.
- Immunoprecipitate using 1 mg of lysate protein (may have to use more depending on the protein you’re looking at) mixed with 5 mg of antibody and 10 ml of the washed Protein A Sepharose. Total volume of the mixture should be 1 ml.
- Incubate on a rocker platform at 4ºC for 1 hour.
- Wash 3x with 1 ml of PLC buffer.
- Add 50 ml 1x sample buffer.
- Boil 5 min.
- Load 20 ml on an SDS PAGE gel.
This method is based, with permission, on an original protocol available here.