The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins
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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2006-02-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520336452 |
| _version_ | 1818739628409094144 |
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| author | W. Sean Davidson Amy B. Ghering Lauren Beish Matthew R. Tubb David Y. Hui Kevin Pearson |
| author_facet | W. Sean Davidson Amy B. Ghering Lauren Beish Matthew R. Tubb David Y. Hui Kevin Pearson |
| author_sort | W. Sean Davidson |
| collection | DOAJ |
| description | 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. 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. |
| first_indexed | 2024-12-18T01:27:51Z |
| format | Article |
| id | doaj.art-d5d0ffdcd5a542d5849000914a6c4449 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-18T01:27:51Z |
| publishDate | 2006-02-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-d5d0ffdcd5a542d5849000914a6c44492022-12-21T21:25:41ZengElsevierJournal of Lipid Research0022-22752006-02-01472440449The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteinsW. Sean Davidson0Amy B. Ghering1Lauren Beish2Matthew R. Tubb3David Y. Hui4Kevin Pearson5Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507The 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. 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.http://www.sciencedirect.com/science/article/pii/S0022227520336452lipid binding assayfluorescencesmall unilamellar vesiclebiotin/streptavidin |
| spellingShingle | W. Sean Davidson Amy B. Ghering Lauren Beish Matthew R. Tubb David Y. Hui Kevin Pearson The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins Journal of Lipid Research lipid binding assay fluorescence small unilamellar vesicle biotin/streptavidin |
| title | The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins |
| title_full | The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins |
| title_fullStr | The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins |
| title_full_unstemmed | The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins |
| title_short | The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins |
| title_sort | biotin capture lipid affinity assay a rapid method for determining lipid binding parameters for apolipoproteins |
| topic | lipid binding assay fluorescence small unilamellar vesicle biotin/streptavidin |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520336452 |
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