Expression and purification of recombinant annexin V for the detection of membrane alterations on apoptotic cells.
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 4microg 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.
Brumatti
Gabriela
G
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Sheridan
Clare
C
Martin
Seamus J
SJ
D000818:Animals; D017304:Annexin A5 / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D017209:Apoptosis; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D008856:Microscopy, Fluorescence; D010718:Phosphatidylserines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013194:Staining and Labeling
D017304:Annexin A5; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D016650:Fluorescein-5-isothiocyanate