Stable isotope labeling by amino acids in cultured primary neurons

Cultured primary neurons are a well-established model for the study of neuronal function. Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires nearly complete metabolic labeling of proteins and therefore is difficult to apply to cultured primary neurons, which do not divide in culture. Here we describe a protocol that utilizes a multiplex SILAC labeling strategy for primary cultured neurons. In this strategy, two different sets of heavy amino acids are used for labeling cells for the different experimental conditions. This allows for a straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled.