The biotin-capture lipid affinity assay: A rapid method for determining lipid binding parameters for apolipoproteins

W. Sean Davidson, Amy B. Ghering, Lauren Beish, Matthew R. Tubb, David Y. Hui, Kevin Pearson

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. Mutagenesis techniques have been widely used to modulate apolipoprotein lipid affinity for studying biological function, but the approach requires rapid and reliable lipid affinity assays to compare the mutants. Here, we describe a novel method that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUVs) containing trace biotinylated and fluorescent phospholipids. After a 30 min incubation at various apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted and measured in a fluorescence microplate reader for calculation of a dissociation constant and the maximum number of potential binding sites on the SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV, and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations.jlr The assay is easy to perform and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 h, consuming only 120 μg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.

Original languageEnglish
Pages (from-to)440-449
Number of pages10
JournalJournal of Lipid Research
Volume47
Issue number2
DOIs
StatePublished - Feb 2006

Keywords

  • Biotin/streptavidin
  • Fluorescence
  • Lipid binding assay
  • Small unilamellar vesicle

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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