Part of the long blacklist of proteins that repeatedly gatecrash IP experiments.

Like gatecrashers at a celebrity party, there are proteins that turn up in immunoprecipitation (IP) experiments despite having no real association with the A-list protein of interest. But now, researchers-cum-bouncers, Trinkle-Mulcahy et al. are armed with a list of repeat gatecrashers to look out for.

One way to lessen the chance of uninvited proteins turning up in your IP is to increase the stringency of purification methods; however, this is also likely to remove low affinity, low abundance and yet genuine interaction partners. To keep stringency low but cope with the large number of co-precipitating proteins, researchers have developed a mass spectrometry approach called SILAC (stable isotope labeling with amino acids in culture) that identifies and quantifies all precipitated proteins.

Trinkle-Mulcahy and colleagues now describe an optimized methodology for the technique with the important addition of a “bead proteome”—a blacklist of proteins that bind nonspecifically to the three most commonly used affinity matrices: magnetic, sepharose, and agarose beads. The authors identified the culprits by examining all proteins that repeatedly precipitated at similar quantities in test and control samples.

If a protein appears on the list, it should not be automatically discounted—these gatecrashers have parties at which they are genuine invitees. And of course, proteins not on the list should not be assumed to be bona fide, either. The authors have so far compared commonly observed nonspecific binding proteins for two different cell types, three different matrices, and one affinity tag, and so are quick to point out that the blacklist is only a guide. That said, with the time and expense that goes into confirming possible interaction partners, the blacklist now enables researchers to focus their attentions on the most likely VIPs of IP.

Trinkle-Mulcahy, L., et al.
J. Cell Biol.