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.

ABSTRACT: BACKGROUND:The removal of high-abundance proteins from plasma is an efficient approach to investigating flow-through proteins for biomarker discovery studies. Most depletion methods are based on multiple immunoaffinity methods available commercially including LC columns and spin columns. Despite its usefulness, high-abundance depletion has an intrinsic problem, the sponge effect, which should be assessed during depletion experiments. Concurrently, the yield of depletion of high-abundance proteins must be monitored during the use of the depletion column. To date, there is no reasonable technique for measuring the recovery of flow-through proteins after depletion and assessing the capacity for capture of high-abundance proteins. RESULTS:In this study, we developed a method of measuring recovery yields of a multiple affinity removal system column easily and rapidly using enhanced green fluorescence protein as an indicator of flow-through proteins. Also, we monitored the capture efficiency through depletion of a high-abundance protein, albumin labeled with fluorescein isothiocyanate. CONCLUSION:This simple method can be applied easily to common high-abundance protein depletion methods, effectively reducing experimental variations in biomarker discovery studies.

SUBMITTER: Kim K 

PROVIDER: S-EPMC3012035 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature