Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 309

2. Add 1/5th volume 100% trichloroacetic acid (TCA; 200 m L).
3. Leave samples at 4 C overnight.
4. Spin for 30 min at maximum speed in microcentrifuge. Remove supernatant and
spin again for 1 min. Remove any residual supernatant with gel loading tip,
leaving 5-10 m L behind.
5. Wash twice with 500 m L cold acetone. Spin 10 min at maximum speed in
microcentrifuge.
6. Dry the protein pellet by spinning briefly in a speed vac.
7. Freeze in liquid nitrogen and store at 80 C. The protein pellet is suitable for
direct mass spectrometric analysis.
8. After elution with glycine, the beads should be boiled in Sample Buffer and
analyzed by silver staining/immunoblotting to assess elution efficiency.
E. Urea Elution: For Mass Spectrometry
Urea elution can also be used for mass spectrometry. In practice, we find that
glycine elution works better for elution of the antigen from the antibody and for
detection of the purified antigen in mass spectrometric analysis.
1. After standard immunoprecipitation (see Section VI.B), wash beads with Pre-
urea Wash Buffer. Remove all residual supernatant.
2. Add 75 m L Urea Elution Buffer and rotate for 30 min at RT.
3. Pellet beads and transfer eluate to a new tube. Re-pellet to ensure removal of all
protein A beads.
4. Remove 50 m L of elution and drop freeze in liquid nitrogen to send for mass
spectrometry. Add Sample Buffer to the rest to run on a gel.
VII. Tandem Affinity Purification Using a LAP Tag
As a first step, the extract is incubated with anti-GFP antibody-coupled
protein A beads ( Fig. 3A ). For this purpose, we use in-house rabbit polyclonal
anti-GFP antibodies generated by injecting purified GFP in rabbits.
Recombinant GFP-binding domains from single-chain antibodies have also been
used successfully for affinity purification of GFP-tagged proteins ( Trinkle-
Mulcahy et al., 2008 ). After immunoprecipitation of GFP, the fusion protein
is released by TEV protease cleavage ( Fig. 3A ). The subsequent purification on
S Protein agarose further enriches for complexes containing the fusion protein.
Extract prepared from an untagged strain can serve as negative control for the
TAP procedure.
A. TEV Cleavage
Perform immunoprecipitation using anti-GFP antibody-coupled beads as
described in Section VI.B.