Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.
Materials and Methods
SILAC and preparation
NP-40 Buffer (for 200 ml)
Stock concentration | Add | |
10 mM sodium phosphate buffer (pH 7.2) | 0.1 M | 20 ml |
150 mM NaCl | 2 M | 15 ml |
1% NP-40 | 100% | 2 ml |
50 mM NaF | 1 M | 10 ml |
0.1 mM Na3VO4 | 10 mM | 2 ml |
Volume to 200 ml |
ADD before use:
TEV-C Buffer (for 50 ml)
Stock concentration | Add | |
25 mM Tris (pH 8.0) | 100 mM | 12.5 ml |
150 mM NaCl | 2 M | 3.75 ml |
0.1 % NP-40 | 100 % | 50 _l |
0.5 mM EDTA | 500 mM | 50 __l |
Volume to 50 ml |
ADD before use:
The aliquots saved during the purification procedure should include (1) pre-cleared cell lysate, (2) bound fraction, (3) unbound fraction, and (4) eluted fraction. We recommend analyzing the protein contents of the above aliquots by Western blotting using anti-TAP antibody or silver staining to reflect the binding and eluting efficiency of the experiment. Examples of a silver stained gel and a blot are shown in Figure 1.
Since SILAC provides us with unbiased and quantified measurements of protein binding partners, we only do the first stage of the TAP purification to minimize sample loss. If you experience significant amounts of unspecific binding, you may wish to carry out the entire protocol, which can be found in the Cold Spring Harbor manual (4).