Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain
Adapted from Live Cell Imaging, 2nd edition (ed. Goldman et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2010.INTRODUCTION
Visualization of a certain lipid species can be achieved by producing a fusion protein between a lipid-binding domain and a visible fluorescent protein (VFP). After a DNA construct for a VFP-tagged lipid-binding domain has been prepared, the desired cell line is transfected with the DNA and visualized using fluorescence microscopy, as described here. The DNA encoding a VFP-tagged lipid-binding domain is isolated from Escherichia coli, and the cells to be transfected are grown on glass-bottom dishes or on coverslips. We routinely perform transfection with commercially available reagents, although, depending on the cell line, the calcium phosphate precipitation method may provide an economic alternative.
