Friday, September 23, 2016
iMixPro -- less false discoveries in pulldowns with heavy peptides!
Affinity purifications (or much cooler...affinity enrichments!) are ever in increasing demand. What protein interacts with my other proteins and how may be one of the most important things we'll be contributing to biology in the future -- once its not completely fracking impossible to do it. The new crosslinking methodologies that are coming are going to help, but iMixPro is another elegant approach.
It is described in this awesome new JPR paper from Sven Eyckerman et al.,! I'll start off by saying it isn't the simplest method you've ever seen, but if you've spent much time doing protein-protein interaction assays you've either developed your own complex methodology that works and you're keeping it secret from the world -- or you'd try just about anything to figure out what is real and what is not! -- especially when today's super sensitive instrumentation is telling you you pulled down 1,000 proteins with that expensive "specific" antibody you just got in!
It differs from affinity enrichments in that it intelligently employs heavy labels (this is where the "i" comes from in the name). Having essentially SILAC pairs to look at in their data improves even the label free quan approach you have in affinity enrichments. Combining labeled peptides = less batch effects, which is never a bad thing. They show some great examples where they can remove the noise and find their true interactors, even when the intensity of the true signal is only a fraction of the value of the other signals identified!