Figure 1From: Online monitoring of immunoaffinity-based depletion of high-abundance blood proteins by UV spectrophotometry using enhanced green fluorescence protein and FITC-labeled human serum albumin Schematic of experiments. (A) In the first stage, EGFP was purified, and the correlation between the quantity of EGFP and UV absorbance at 488 nm was calculated. From these data, a standard curve was generated. (B) 300 μg EGFP was spiked into 2.21 mg of 5-fold-diluted plasma, and MARS depletion was performed under UV488 nm monitoring in 6 repeat runs. Unbound and bound fractions were analyzed by SDS-PAGE and 2-DE. In addition, Western blot was performed for EGFP, alpha-1-antitrypsin, transferring, and haptoglobin. The OD at 488 nm was measured to determine the recovery of EGFP from EGFP-only and EGFP-spiked plasma. (C) During 200 runs of the MARS column, EGFP-only or EGFP-spiked plasma was injected as an indicator of flow-through proteins for the quality assessment at every 20th run, in which the reproducibility of depletion was calculated. (D) Depletions were performed using FITC-HSA as a high-abundance protein indicator to determine the capture efficiency of high-abundance proteins.Back to article page