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Figure 1 | Proteome Science

Figure 1

From: 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

Figure 1

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.

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