IPG strips were purchased from Amersham Biosciences (San Francisco, CA, USA) and proteins were isoelectrofocused using the PROTEAN IEF Cell™ (Bio-Rad, Hercules, CA, USA) IEF system, essentially as described by Go"rg et al. . Briefly, samples were thawed and diluted into IEF sample buffer containing 9 m urea, 2 m thiourea, 4% CHAPS, 0.1 m DTT, 0.8% pH 3-10 linear carrier ampholyte buffer to yield the desired protein amount in a volume that could be absorbed by the IPG strip used (typically 400 mL for an 18cm IPG strip). IPG strips (typically 18cm) were rehydrated with sample buffer containing serum proteins at 50 V for 12 h and then the applied voltage was increased in a linear fashion to a maximum of 10 000 V until a total of 60000 Vh was reached. The IPG strips were equilibrated and applied to 18cmx 19 cm, 1.0 mm thick second dimension 10% Tris-tricine polyacrylamide gels , cast without stacking gels and with sodium thiosulfate added to reduce silver stain background, as described by Hochstrasser et al. . Gels were run at 100 mA constant current with external cooling (~4h) until the tracking dye migrated to within 1 cm of the bottom of the gel. Either colloidal CBB or Silver Quest (Invitrogen) was used to visualize proteins after 2-DE. For protein identification, protein spots were excised using biopsy punches (Miltex Instruments, Lake Success, NY, USA) and subjected to in-gel trypsin digestion. Protein identifications of the 2-D gel spots were performed using an LTQ linear IT mass spectrometer (Thermo Electron, Marietta, OH, USA). The MS/MS spectra were searched against the NCBI nonredundant database, and each assignment was manually validated.
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