Brumatti Gabriela, Sheridan Clare, Martin Seamus J
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Methods. 2008 Mar;44(3):235-40. doi: 10.1016/j.ymeth.2007.11.010.
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.
细胞凋亡是一种细胞死亡方式，其伴随着质膜的特定改变，这些改变促进吞噬细胞对这些细胞的识别和吞噬。尽管已经确定了几种这样的膜改变，但磷脂酰丝氨酸从质膜内小叶向外小叶的重新分布已成为哺乳动物中凋亡细胞最广泛使用的标志物之一。这主要是由于以磷脂酰丝氨酸结合蛋白膜联蛋白V的形式存在用于此事件的灵敏且特异的探针。在此，我们描述了从大肠杆菌中表达和纯化重组多组氨酸标签膜联蛋白V的方法。重组膜联蛋白V高度可溶，因此易于表达并高产纯化；通常每毫升细菌培养物中可获得约4微克蛋白质。我们还描述了将该蛋白与异硫氰酸荧光素荧光团偶联的方法，以及其用于通过流式细胞术或荧光显微镜检测凋亡细胞的方法。
