title: Purification of annexin V and its use in the detection of apoptotic cells.
abstract: Cell death by apoptosis has been studied for many years using fluorescently labeled annexin V. Annexin V shows high affinity for the phosphatidylserine that becomes enriched in the outer leaflet of the plasma membrane during apoptosis, but not necrosis, allowing differentiation between the two types of cell death. In this chapter we detail two methods for the purification of annexin V. The first is an untagged recombinant protein using a three step Fast Protein Liquid Chromatography (FPLC) method, and the second using a single step purification protocol via a glutathione S-transferase (GST) tag. Labeling of the resulting annexin V with a fluorescent dye to allow visualization of the protein is also explained. Finally, two methods are described in which a fluorescently labeled derivative of annexin V is used to detect apoptosis, namely the in vitro method of fluorescence-activated cell sorting (FACS) where fluorescent annexin V is used to differentiate apoptotic and necrotic cells within a population; and detection of apoptosing retinal cells (DARC) allowing the identification of apoptotic cells in the retina in vivo.
authors.affiliation: Division of Cell Biology, Institute of Ophthalmology, University College London, London, UK.
authors.forename: Katy M
authors.initials: KM
authors.lastname: Coxon
authors.forename: James
authors.initials: J
authors.lastname: Duggan
authors.forename: M Francesca
authors.initials: MF
authors.lastname: Cordeiro
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Moss
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017209:Apoptosis; D002457:Cell Extracts; D005591:Chemical Fractionation / Q000379:methods*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000254:growth & development; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000302:isolation & purification* / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000166:cytology; D013997:Time Factors
chemical_list: D017304:Annexin A5; D002457:Cell Extracts; D005456:Fluorescent Dyes; D005982:Glutathione Transferase