title: Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6.
abstract: Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.
authors.affiliation: PerkinElmer Life and Analytical Sciences, Wallac Oy, P.O. Box 10, FIN-20101, Turku, Finland. jarkko.karvinen@perkinelmer.com
authors.forename: Jarkko
authors.initials: J
authors.lastname: Karvinen
authors.forename: Annika
authors.initials: A
authors.lastname: Elomaa
authors.forename: Maija Liisa
authors.initials: ML
authors.lastname: Mäkinen
authors.forename: Harri
authors.initials: H
authors.lastname: Hakala
authors.forename: Veli Matti
authors.initials: VM
authors.lastname: Mukkala
authors.forename: Jari
authors.initials: J
authors.lastname: Peuralahti
authors.forename: Pertti
authors.initials: P
authors.lastname: Hurskainen
authors.forename: Jari
authors.initials: J
authors.lastname: Hovinen
authors.forename: Ilkka
authors.initials: I
authors.lastname: Hemmilä
mesh_terms: D020170:Caspase 1 / Q000032:analysis*; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases / Q000032:analysis*; D002614:Chelating Agents; D005453:Fluorescence; D028581:Lanthanoid Series Elements; D013057:Spectrum Analysis / Q000379:methods; D013379:Substrate Specificity
chemical_list: D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases; D020170:Caspase 1