Biomedical Engineering Reference
In-Depth Information
7. The use of a column ensures that all protein A agarose par-
ticles (together with the “sticky” proteins bound to them)
are removed. A sample from the mixed SILAC lysates can
be used as a quality control for checking by mass spectrom-
etry the exact mixing ratio.
8. For approximately 1.2
10
8
10
7
×
cells (4
×
cells per condi-
tion) we recommend to use 75
μ
g anti-EGFR antibody for
immunoprecipitation.
9. If the mixing ratio differs a normalization coefficient has to
be calculated. This coefficient should be used for normal-
izing all other ratios. These results can be double checked
by analyzing the saved lysate (
see
Note 4
).
10. Quantitation errors are usually in the range of 10-20%.
Specific binding partner should have protein ratios greater
than three times standard deviation.
11. The SILAC approach can be used in virtually every type of
interaction study for unambiguous identification of inter-
action partners. This includes standard protein-bait inter-
action assays such as GST pull down, affinity purifica-
tion of overexpressed tagged proteins or of endogenous
complexes, and protein-peptide interaction assays
(Table
the interaction assay is performed using knockout/RNAi
knockdown/mutant versus the wild-type cells or tissues
Acknowledgments
We thank all CEBI group members for helpful discus-
sions and support. The research leading to these results
has received funding from the European Commission's 7th
Framework Programme (grant agreement HEALTH-F4-2008-
201648/PROSPECTS), the Danish Natural Science Research
Council, the Danish Medical Research Council, and the
Lundbeck Foundation. JD was supported by the European
Molecular Biology Organization and by the Excellence Initiative
of the German Federal and State Governments.
References
1. Gingras, A. C., Gstaiger, M., Raught, B.,
and Aebersold, R. (2007) Analysis of protein
complexes using mass spectrometry.
Nat.
Rev. Mol. Cell. Biol.
8
, 645-654.
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