Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 304

Fig. 7 Experimental outline for protein complex purification. Roman numerals indicate the corre-
sponding sections. A crude extract is generated by grinding and sonicating the worms and embryos
(Section V.1.-4.). Two consecutive centrifugations clear the extract from debris and membranes (Section
V.5.-6.). Antibodies are covalently coupled to beads (Section VI.A.) and extract is incubated with
antibody-coupled beads (Section VI.B.). Protein complexes are eluted with glycine (Section VI.D.1.)
and precipitated with TCA (Section VI.D.2.-7.). Prior to mass spectrometry the eluate is proteolytically
digested (Section VIII.). (For color version of this figure, the reader is referred to the web version of this
book.)
increase. A test sonication should be performed to define conditions at which one
or both of these values begin to plateau.
5. Transfer crude extract to TLA100.3 tube and spin at 20,000g for 10 min at 2 C,
with medium deceleration. Save 20 m L sample.
6. Transfer supernatant to new tube and pellet at 100,000g for 20 min at 2 C. Try to
avoid lipid and re-pellet if too cloudy. Save 20 m L sample.
7. Transfer supernatant to a tube on ice. This extract will be used for purifications.
All samples and the extract may be frozen in liquid nitrogen and stored at 80 C
until further use.
8. Use Bradford reagent to measure protein concentration in extract.