MS Protocols - Interactomics
Identifying interaction partners of proteins is a very powerful proteomics application. Often the bait is expressed as a fusion protein containing an affinity tag for enrichment. We recommend to carry out quantitative experiments in which the bait is isolated under mild conditions to preserve weak interactions. This usually results in the identification of hundreds of proteins of which only few bind specifically the bait protein. All others bind non-specifically to the antibody-coupled beads both in the bait and control reactions and are used for robust normalization. A more detailed description of this quantitative approach can be found here.
Special care has to be taken in designing these AP-MS experiments. It's particularly important that:
- the bait is expressed at endogenous levels.
- bait and control experiments are carried out side-by-side.
- at least three replicates are analyzed to obtain robust statistics.
- final peptides are free of detergents and salts.
Unless you use the protocols provided below, MS samples have to be prepared via in-gel digest to prevent contamination by detergents and salts.
AP-MS: GFP pull-down, Human cell lines, LFQ and SILAC) - Publication: Hubner et al.
AP-MS: Strep-Peptide pull-down using filter plates; mouse tissue; LFQ and SILAC - Publication: Eberl et al.
AP-MS: GFP pull-down, Yeast grown in 96 well plates - Publication: Keilhauer et al.
IP-MS: Flag pull-down from yeast - Tamara Flohr
In-gel digest, FASP, Stage-Tipping, SCX Fractionation, High pH Fractionation, BCA Assay, LFQ, SILAC.