Membrane rafts help coordinate protein trafficking patterns, signaling cascades and steps in viral pathogenesis. Stabilized by cholesterol and the long, saturated, acyl chains of sphingolipids, these liquid-ordered supramolecular domains of plasma membranes and other membranes segregate highly specific subsets of membrane proteins. The structural reality of rafts is no longer in doubt, but the precise roles they play remain an area of controversy. Although the field has incorporated new biophysical and cellular imaging approaches, biochemical isolation of rafts has relied on the now classic observation that raft membranes remain insoluble at 4°C in non-ionic detergents such as Triton X-100. Although instrumental in establishing raft properties, various problems compromise the utility of this method. These include artifactual modification of lipid-phase behavior at low temperatures, significant solubilization of the liquid-ordered domain and loss of proteins with low-affinity association with raft lipids.Now, Hai-Tao He and collaborators [1xTCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts. Drevot, P. et al. EMBO J. 2002; 21: 1–10Crossref | PubMed | Scopus (252)See all References][1] find that a polyoxyethylene ether detergent in the Brij series circumvents these problems and use this detergent to gain a new perspective on T-cell activation. They reasoned that the bulky headgroup and mono-unsaturated ether chain of Brij 98 would cause the detergent to solubilize the fluid phase of the membrane but leave the more tightly packed liquid-ordered phase intact. The hunch proved correct: membrane remaining insoluble at 37°C in the presence of 1% Brij 98 displays the characteristic properties of rafts, including enrichment in sphingomyelins, cholesterol, GPI-anchored, palmitoylated and myristoylated proteins and the absence of prenylated proteins.Previous work on T-cell activation pointed to rafts as dynamic centers that collect signaling components from the surrounding liquid-disordered phase, with the process initiated by T-cell receptors binding to cognate peptide-presenting MHC molecules and repartitioning into rafts. However, a different scenario emerges when rafts are isolated at 37°C. Signaling complexes are found fully primed in the liquid-ordered phase; rafts from cells that have not been activated contain essential signaling components, including the T-cell receptor–CD3 complex, the Lck and ZAP-70 kinases, and the CD4 co-receptor. Moreover, when such rafts are activated and supplied with ATP, appropriate initial phosphorylation events take place. The method also resolves a microheterogeneity among the rafts, with antibodies to Thy-1 precipitating a vesicle population distinct from that containing the signaling machinery. Preliminary data suggest that these populations interact upon T-cell activation. Perspectives opened by this new wrinkle on raft isolation extend within immunology (e.g. does CD8 also associate with the T-cell receptor before activation?) and well beyond.