Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 310

Note: At this stage, it is possible to elute bound material using glycine as described
in Section VI.D. and compare the elution of the one-step GFP immunoprecipitation
to the two-step LAP.
1. Pool beads into a single tube and fill with Lysis Buffer (with 300 mM KCl, 0.5
mM DTT).
2. Add 30 m L of purified TEV protease (1 mg/mL) and rock tubes for 4 h at 4 C.
Add an additional 30 m L of TEV and rock tubes overnight.
3. Pellet beads and transfer supernatant to a new tube. Add 350 m L Lysis Buffer to
beads to remove any residual cleaved protein.
B. S Protein Agarose
1. Wash tube of 85 m L S protein agarose three times with 1 mL Lysis Buffer (with
300 mM KCl).
2. Add TEV protease eluted supernatant to S protein agarose and rock for 3 h at
4 C.
3. Pellet beads and wash three times with Lysis Buffer (with 300 mM KCl).
4. Wash one time with Lysis Buffer with 100 mM KCl without detergent (NP-40).
C. Sample Buffer Elution: For Silver Staining & Immunoblotting
Perform Sample Buffer Elution as described in Section VI.C.
D. Urea Elution: For Mass Spectrometry
Perform Urea Elution as described in Section VI.E.
VIII. Mass Spectrometry & Prioritization for Follow-up
Experiments
Protein mass spectrometry has made remarkable advances in the recent decade.
We will not discuss the details of the methodology, which are extensively reviewed
elsewhere ( Cravatt et al., 2007; Yates et al., 2009 ). We typically do not separate
proteins from immunoprecipitates on gels prior to mass spectrometry. Instead, the
entire eluate is proteolytically digested and the peptide mixture is separated by
multidimensional liquid chromatography. The mass/charge ratio of ionized peptides
is determined in the first mass analyzer, and then the peptides are fragmented in the
collision cell and passed through the second mass analyzer to determine amino acid
sequence. As mentioned above, current methods can yield up to 1000 proteins in
one-step immunoprecipitation performed using an antibody that passes generally
accepted antibody specificity criteria. This abundance of information makes it