Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasma α1-Acid Glycoprotein
Purification of plasma AGP from pooled human plasma. (a) Ion-exchange chromatography of human plasma on a DEAE-Sepharose FF column (10 × 50 cm) equilibrated with 0.02 M citrate phosphate buffer, pH 4.0. Fifty mL of the dialyzed plasma sample was applied to the column. Fractions were assayed for protein (●) and AGP concentrations. AGP appeared only in the fractions eluted with 0.02 M citrate-phosphate buffer, pH 7.0 containing 0.2 M NaCl (—). (b) Ion-exchange chromatography of the eluate from DEAE-Sepharose FF column on an SP-Sepharose FF column (2 × 20 cm) equilibrated with 0.02 M citrate-phosphate buffer, pH 4.0. Ten mL of the pooled and dialyzed samples were applied to the column. Fractions were assayed for protein (●) and AGP concentration. AGP appeared in the eluate of citrate-phosphate buffer at pH 4.8 (—). (c) 10/20 SDS-polyacrylamide gel electrophoresis of purified AGP preparations. Lane 1: pooled plasma sample; lane 2: DEAE-Sepharose FF eluted fractions (0.2 M NaCl, pH 7.0); lanes 3, 4: DEAE- (0.2 M NaCl, pH 7.0) and SP- (pH 4.8) Sepharose FF eluted fractions. After electrophoresis, the gel was blotting on the membrane and each lane on the membrane was stained with Coomassie Brilliant Blue (lanes 1–3) and anti-human AGP antibody (lane 4). See the details in the Text.
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