Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6.
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.
Karvinen
Jarkko
J
PerkinElmer Life and Analytical Sciences, Wallac Oy, P.O. Box 10, FIN-20101, Turku, Finland. jarkko.karvinen@perkinelmer.com
Elomaa
Annika
A
Mäkinen
Maija Liisa
ML
Hakala
Harri
H
Mukkala
Veli Matti
VM
Peuralahti
Jari
J
Hurskainen
Pertti
P
Hovinen
Jari
J
Hemmilä
Ilkka
I
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
D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases; D020170:Caspase 1