Detection of calcium binding proteins on polyacrylamide gels using time-resolved lanthanide luminescence photography.
Methods were developed for using the luminescent lanthanides Tb3+ and Eu3+ for the specific staining of calcium-binding proteins, as well as the nonspecific staining of proteins, on polyacrylamide gels. These methods involve equilibration of the gel after electrophoresis in solutions containing the appropriate lanthanide and a weak competitive chelating agent, such as N-(2-hydroxyethyl)iminodiacetic acid or nitrilotriacetic acid. This staining has the potential for complete reversibility using stronger chelating agents such as EDTA or diethylenetriaminepentaacetic acid, to allow for recovery of the protein. Specific staining produces an intense luminescent signal from those metal-binding proteins which have been modified either chemically or via site-directed mutagenesis. Gels were photographed using a time-resolved fluorescence camera system.
Hill
I E
IE
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
Hogue
C W
CW
Clark
I D
ID
MacManus
J P
JP
Szabo
A G
AG
D000180:Acrylic Resins; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000235:genetics; D002614:Chelating Agents; D005063:Europium; D008163:Luminescent Measurements; D008674:Metals, Rare Earth; D009154:Mutation / Q000235:genetics; D010781:Photography / Q000379:methods*; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling / Q000379:methods; D013725:Terbium
D000180:Acrylic Resins; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D008674:Metals, Rare Earth; C016680:polyacrylamide gels; D013725:Terbium; D012967:Sodium Dodecyl Sulfate; D005063:Europium