Q1: Can anyone do a SILAC IP experiment?

A: If you can culture cells, do an IP and run a gel, you can do a SILAC IP experiment! Your cells are labeled metabolically, meaning that all you have to do is grow them in the appropriate tissue culture media (For details on sourcing reagents and preparing the media, download our SILAC Reagent Protocol.) This media is very easy to prepare. You can then prepare whole cell lysates/extracts or fractionate the cells however you wish (For details on cell fractionation and preparation of whole cell vs. nuclear and cytoplasmic lysates, download our Cell Fractionation Protocol.) The IP is your standard IP, followed by a 1D SDS-PAGE gel to separate the proteins. You can either digest them yourself (For details on trypsin digestion of gel slices for MS analysis, download our In Gel Digestion Protocol.) or have your mass spec facility do the digestions for you. The samples can be run by your favorite mass spec facility, and the final data quantified using the freely available open source MSQuant software.

Q2: How expensive is a SILAC IP experiment?

A: Most of the cost of the experiment lies in the mass spec analysis, and there are ways to minimize cost throughout the experiment. You will need custom DMEM in which Arg and Lys have been left out, dialyzed fetal calf serum, and both standard powdered Arg and Lys (for R0K0 media) and the isotopic Arg and Lys (For details on sourcing reagents and preparing the media, download our SILAC Reagent Protocol.). A good way to minimize cost of the isotopes is to buy in bulk, i.e. sharing an order between more than one lab, and to negotiate bulk discounts with the companies.

Q3: Can I use the SILAC approach for endogenous IPs?

A: Yes you can. The simplest experiment is a comparison of an IP using purified IgG (cells grown in R0K0) to an IP of your protein of interest (cells grown in R6K6) using a specific antibody. The important thing is to covalently bind both antibodies at the same concentration (e.g. 1 mg/ml), IP from lysates with the same total protein concentration, and carefully wash and combine the beads so that none are lost. All proteins are then eluted from the combined beads and run in a single lane on a gel. If this is done properly, your pre-immune IP acts as your built-in negative control, and any contaminating proteins that stick to the beads will have a SILAC ratio of 1:1. You can also use triple encoding SILAC to look at your protein of interest under two different conditions (e.g. negative control plus IP protein from untreated cells and IP protein from cells perturbed in some way, i.e. exposed to DNA damage or transcriptional inhibition).

These are the most commonly asked questions. We'll continue to add to this FAQ as people approach us with more questions!

SILAC Protocols:

For details on sourcing reagents and preparing the media, download our SILAC Reagent Protocol.

For details on covalently coupling antibodies to Protein G sepharose, download our Covalent Coupling Protocol.

For details on cell fractionation and preparation of whole cell vs. nuclear and cytoplasmic lysates, download our Cell Fractionation Protocol.

For details on trypsin digestion of gel slices for MS analysis, download our In Gel Digestion Protocol.