Figure 4

Performance of EGFP from EGFP-only or EGFP-spiked plasma in depletion experiments. (A) Overlay of chromatograms from 6 consecutive MARS depletion runs using EGFP-spiked plasma. A mixture of flow-through proteins and EGFP as a flow-through protein indicator were eluted as an unbound fraction at 17 min, and the high-abundance proteins were bound to the MARS column and eluted as the bound fraction at 35 min. (B) Thirty micrograms of bound fraction (high-abundance proteins) was subjected to 2-DE for 7 cm from pH 4 to pH 7. Six high-abundance proteins on a silver-stained gel were visualized, and MALDI-TOF/TOF analysis was performed to identify spots (data not shown). (C) Equal amounts (5 μg) of plasma (P), unbound fraction (UB, flow-through proteins), and bound fraction (B, high-abundance proteins) from the EGFP-spiked plasma depletion were run on an 8-12% SDS-PAGE gel and visualized by silver staining. The thick band at 25 kDa in the UB lane constitutes spiked EGFP; there is no such band in the B lane. (D) The unbound fraction (UB) and bound fraction (B) were analyzed by Western blot to detect target proteins. Anti-6×His tag was used to detect His-tagged EGFP, and antibodies for antitrypsin, haptoglobin, and transferrin were used for the bound and unbound fractions from the 6 consecutive depletion runs. Serum albumin and immunoglobulin were excluded from the Western blot due to their abundance. (E) Each protein in the Western blots was expressed as band intensity and shown on a bar graph with standard errors, using the Phoretix gel analysis program. In the alpha-1-antitrypsin and transferrin results, there were traces of the unbound fraction, which might have been caused by homology to the serpine family and other proteins.