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.
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.


CONCLUSIONS
This simple method can be applied easily to common high-abundance protein depletion methods, effectively reducing experimental variations in biomarker discovery studies.
Department of Biomedical Sciences, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.
Kyunggon
K
Kim
Department of Biomedical Sciences, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.
Jiyoung
J
Yu
Department of Biomedical Sciences, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.
Hophil
H
Min
Department of Biomedical Sciences, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.
Hyunsoo
H
Kim
Department of Biomedical Sciences, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.
Byungwook
B
Kim
Department of Ophthalmology, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.
Hyeong Gon
HG
Yu
Department of Biomedical Sciences, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.|Department of Biomedical Engineering, Seoul National University, College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.|Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-Dong, Seoul 110-799, Korea.
Youngsoo
Y
Kim