Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 303

13. Resuspend pellet in 500 m L Nuclei Buffer (with 0.1% digitonin and protease
inhibitors) and mix with 5 mL 30% (w/v) sucrose cushion in Nuclei Buffer with
0.1% digitonin.
14. Centrifuge at 2000g for 15 min. The pellet will be enriched for nuclei.
15. Check integrity of the nuclei under a microscope after incubation with 1 m g/mL
Hoechst stain ( Fig. 6C ).
V. Preparing Worm and Embryonic Extract
Extract can be prepared using whole worms harvested from liquid culture or from
embryos isolated by bleaching. Lysis of worms and embryos is performed in an
isotonic buffer. Subsequent to lysis, the salt concentration can be raised to 300 mM
KCl to enhance stringency of protein complex isolation. Worms and embryos are
lysed by grinding in liquid nitrogen followed by sonication ( Fig. 7 ). Two consecutive
centrifugations remove membranes and lipids and the supernatant is used for com-
plex isolation. The lysis conditions described here are not well-suited for membrane-
associated proteins. A protocol for isolating membrane proteins is described in
Gottschalk et al. (2005) .
1. Pre-cool a mortar and pestle by filling with liquid nitrogen for at least 5 min.
2. Weigh out frozen adult/embryo beads. For one immunoprecipitation use 1gof
frozen worm/embryo beads. Grind frozen beads to a fine powder: initially break
down worm/embryo beads by gentle tapping (try not to lose too many beads as
they have a tendency to jump out of the mortar), then grind. Keep mortar cold by
adding more liquid nitrogen as necessary and waiting for it to evaporate. For more
than 40 g of worms, one can also use a warring blender cooled with liquid
nitrogen. For embryo extracts, the freeze-grinding step may be skipped, as son-
ication is sufficient to release cytoplasmic contents.
3. Add an equal volume of 1.5 Lysis Buffer (with protease inhibitors) to each gram
of adult/embryo beads. Keep 20 m L sample for gel analysis.
4. Set up ice-water bath and sonicate with a tip sonicator (e.g., Branson Digital
Sonifier). It is critical to prevent heating of the sample during sonication. For a
Branson Digital Sonifier with a microtip use the following settings:
30% amplitude for 3 min total (15 s on; 45 s off - after each 1 min of sonication
wait 2 min to chill)
40% amplitude for 30 s (15 s on; 45 s off) Save 20 m L sample.
We recommend optimizing the sonication protocol by two methods:
Use a dissecting microscope to monitor worm/embryo lysis. At least 80-90% of
the worms/embryos must be lysed.
Use Bradford reagent or UV absorbance to directly monitor lysis. As cells lyse,
the protein concentration and A 260 absorbance (due to nucleic acid release) will