Micro SolIEF fractionation

MicroSol-IEF was performed using a ZOOM IEF Fractionator (Invitrogen, Carlsbad, CA, USA). Depleted plasma was fractionated on a seven-chamber device, separated by immobiline gel membranes having pH values of 3.0, 4.4, 4.9, 5.4, 5.9, 6.4, 8.1, and 10.0. The pH 3.0 and 10.0 membranes were obtained commercially (Invitrogen), while the remaining membranes were prepared as described previously [21]. Reduced and alkylated plasma was diluted to 3.5 mL and adjusted to the same constituent concentrations as the MicroSol buffer, i.e., 8 m urea, 2 m thiourea, 4% CHAPS, 1% DTT, 0.2% carrier ampholytes, pH 3-10L. Aliquots (700 mL) of the sample were loaded into the inner five chambers, and the remaining two outer chambers were filled with MicroSol buffer without sample. Depleted serum was fractionated on a five-chamber device, with pH 3.0, 4.6, 5.4, 6.2, 7.0, and 10.0membranes obtained commercially (Invitrogen). Acetone-precipitated serum was dissolved in 700 mL of MicroSol buffer and was loaded into the central chamber of the device only

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