Expression, purification and use of recombinant annexin V for the detection of apoptotic cells.
Apoptosis is a mode of programmed cell death that is widely used to eliminate cells during development, tissue homeostasis, infection or in response to injury. Alterations to the plasma membranes of apoptotic cells trigger recognition and engulfment of such cells by phagocytes. Measurement of plasma membrane phosphatidylserine externalization, using fluorescently labeled annexin V, is widely used for the detection of apoptotic cells. Here we describe protocols for bacterial expression, purification and FITC labeling of recombinant annexin V. By following the method outlined in this protocol, it is possible to produce milligram amounts of recombinant annexin V within 3 d. We also describe a method for the assessment of annexin V binding to cell populations by flow cytometry or fluorescence microscopy.
Logue
Susan E
SE
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland.
Elgendy
Mohamed
M
Martin
Seamus J
SJ
D017304:Annexin A5 / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D017209:Apoptosis; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods; D010718:Phosphatidylserines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D014169:Transformation, Bacterial
D017304:Annexin A5; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; D016650:Fluorescein-5-isothiocyanate