Dataset Information

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

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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.

Kim Kyunggon K Yu Jiyoung J Min Hophil H Kim Hyunsoo H Kim Byungwook B Yu Hyeong Gon HG Kim Youngsoo Y

Proteome science 20101201

<h4>Background</h4>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 ...[more]

PMID: 21122139

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Project description:Due to the fact that surfactant molecules are known to alter the structure (and consequently the function) of a protein, protein-surfactant interactions are very important in the biological, pharmaceutical, and cosmetic industries. Although there are numerous studies on the interactions of albumins with surfactants, the investigations are often performed at fixed environmental conditions and limited to separate surface-active agents and consequently do not present an appropriate comparison between their different types and structures. In the present paper, the interactions between selected cationic, anionic, and nonionic surfactants, namely hexadecylpyridinium chloride (CPC), hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), polyethylene glycol sorbitan monolaurate, monopalmitate, and monooleate (TWEEN 20, TWEEN 40, and TWEEN 80, respectively) with bovine serum albumin (BSA) were studied qualitatively and quantitatively in an aqueous solution (10 mM cacodylate buffer; pH 5.0 and 7.0) by steady-state fluorescence spectroscopy supported by UV spectrophotometry and CD spectroscopy. Since in the case of all studied systems, the fluorescence intensity of BSA decreased regularly and significantly under the action of the surfactants added, the fluorescence quenching mechanism was analyzed thoroughly with the use of the Stern-Volmer equation (and its modification) and attributed to the formation of BSA-surfactant complexes. The binding efficiency and mode of interactions were evaluated among others by the determination, comparison, and discussion of the values of binding (association) constants of the newly formed complexes and the corresponding thermodynamic parameters (ΔG, ΔH, ΔS). Furthermore, the influence of the structure of the chosen surfactants (charge of hydrophilic head and length of hydrophobic chain) as well as different environmental conditions (pH, temperature) on the binding mode and the strength of the interaction has been investigated and elucidated.

Dataset Information

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.

Publications

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.

Similar Datasets

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