Transfection of cells with DNA encoding a visible fluorescent protein-tagged lipid-binding domain.
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.
Schultz
Carsten
C
Neef
Anne B
AB
Gadella
Theodorus W
TW
Goedhart
Joachim
J
D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA