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Lipid-Sensing High-Throughput ApoA-I Assays.
MedLine Citation:
PMID:  22811478     Owner:  NLM     Status:  Publisher    
Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.
Anita Niedziela-Majka; Latesh Lad; Jeffrey W Chisholm; Leanna Lagpacan; Karen Schwartz; Magdeleine Hung; Debi Jin; Wanchi Fung; Katherine M Brendza; Xiaohong Liu; Nikos Pagratis; Roman Sakowicz
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-7-17
Journal Detail:
Title:  Journal of biomolecular screening     Volume:  -     ISSN:  1552-454X     ISO Abbreviation:  -     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-7-19     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9612112     Medline TA:  J Biomol Screen     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
1Gilead Sciences, Inc., Foster City, CA, USA.
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