IPG strips were purchased from GE Healthcare and proteins were isoelec-trofocused using the PROTEAN IEF Cell™ (Bio-Rad Laboratories) IEF system, essentially as described by Gorg et al. . Frozen samples were briefly thawed and diluted into IEF sample buffer containing 9 m urea, 2 m thiourea, 4% CHAPS, 0.1 m DTT, 1.6% pH 3-10 linear carrier ampholyte buffer to yield 100 mg of protein in 400 mL sample buffer. IPG strips (18 cm) 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 60 000 Vh was reached. The IPG strips were equilibrated and applied to 18 x 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 (~4 h) until the tracking dye migrated to within 1 cm of the bottom of the gel. Gels were then stained with SYPRO Ruby and imaged using the ProXpress Proteomic Imaging System.
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