Figure 3.

2D-gel electrophoresis of T-cell lipid rafts. 50 × 10⁶ Jurkat T cells were extracted in ice-cold lysis buffer containing 1% Triton-X-100 detergent plus protease and phosphatase inhibitors. Lipid rafts were purified by flotation on sucrose gradient and resuspended in rehydration buffer appropriate for isoelectric focusing of proteins on 3–10 pH strips. The rehydration buffer also contained 20 mm MβCD, which assists in the complete disruption of lipid rafts. Following 2D-gel electrophoresis, proteins were visualized with a silver stain compatible for analysis with mass spectrometry. Discernible protein spots were excised, digested with trypsin and the resulting peptides were recorded by mass spectrometry. The peptide ‘fingerprint’ obtained from this analysis was used to search available protein databases. Arrows indicate protein spots for which a positive identification was made and their identity is summarized on Table 1.