Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 301

A.-E. It is important to keep the temperature at or below 16 C to prevent meta-
phase arrest. Bleached larvae are used to inoculate a synchronized liquid culture
(see Section III.F.) and are shifted to 24 C when the majority of worms are at the
L4 stage. The time point of temperature shifting has to be chosen carefully
because an early shift causes larval arrest and a late shift will ''contaminate''
the extract with mitotic embryos. Worms are harvested when the majority contains
4-8 one-cell meiotic-arrested embryos and worm extract is prepared as described
in Section V. Timing is once again critical since after 4-5 h the embryos overcome
the meiotic arrest and start cycling endo-mitotically (DNA is replicated but cell
division does not occur). The quality of the culture can be assayed by cutting 2-3
worms in a drop of L-15 blastomere culture medium ( Edgar, 1995 ) containing
1 m g/mL Hoechst 33342, and analyzing the shape of the chromosomes.
Metaphase I arrested embryos display six maternal chromosomes that have a
typicalovalshape( Fig. 5B ). Endo-mitotic embryos contain decondensed chromo-
somes that have a fibrous-like appearance and tend to detach from the anterior
cortex of the embryo ( Fig. 5B ).
The above two examples are specific to our research interests but related strategies
can be used to enrich for cell types or stages of interest prior to protein complex
isolations.
IV. Isolation of Nuclei from Embryos
If the protein of interest is a nuclear protein or if DNA-protein interactions are
being analyzed as in Chromatin Immunoprecipitation (ChIP) experiments, nuclei
can be isolated from embryos prior to further analysis. Embryos are isolated by
bleaching as described in Section III.E Steps 1-7; however it is important that
embryos are not frozen prior to nuclei isolation. To isolate nuclei, the embryo
eggshell is digested using chitinase ( Fig. 6A ). Dounce homogenization in hypo-
tonic buffer liberates intact nuclei. A 100g centrifugation removes large debris
and a subsequent 2000g centrifugation pellets the nuclei. A final centrifugation
step over a sucrose cushion is used to separate the nuclei from membrane con-
taminants. Enrichment of nuclei can be easily followed by the emergence of the
core histone bands on Coomassie-stained gels ( Fig. 6B ). Because the chitinase
step is performed at room temperature, nuclei must be purified from freshly
isolated embryos to avoid protein degradation. 1 mL of embryo pellet typically
yields 100 m Lofnuclei.
1. Harvest embryos by bleaching as described in Section III.E. until step 7.
2. Add two embryo pellet volumes of Embryo Buffer and 250 m L of chitinase
stock solution per mL of embryo pellet.
3. Rotate at RT for 30 min. Monitor eggshell integrity under dissecting micro-
scope. Embryos without an eggshell will lose their oval shape and fall apart into
clumps of cells.