Journal
METHODS
Volume 44, Issue 3, Pages 235-240Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2007.11.010
Keywords
annexin V; apoptosis; bacterial expression; phosphatidylserine; plasma membrane changes
Funding
- Wellcome Trust Funding Source: Medline
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Apoptosis is a mode of cell death that is accompanied by specific alterations to the plasma membrane that promote the recognition and engulfment of these cells by phagocytes. Although several such membrane alterations have been defined, redistribution of phosphatidylserine from the inner to the outer plasma membrane leaflet has become one of the most widely used markers for apoptotic cells in mammals. This is largely due to the availability of a sensitive and specific probe for this event in the form of the phosphatidylserine-binding protein, annexin V. Here, we describe methods for the expression and purification of recombinant polyhistidine-tagged annexin V from Escherichia coli. Recombinant annexin V is highly soluble and is thus readily expressed and purified to high yields; typically in the region of 4 mu g of protein per ml of bacterial culture. We also describe methods for conjugation of this protein to the FITC fluorophore and for its use for the detection of apoptotic cells by flow cytometry or fluorescence microscopy. (C) 2007 Elsevier Inc. All rights reserved.
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