Sample Files

Caspase-3 Assay (Assay Designs)

November 21, 2012


Related Products: Gen5 for Detection

Gen5 version required: 1.0 or higher

Basis for the Assay:

Caspase proteins are proteolytic enzymes involved in programmed cell death or Apoptosis.
This proteolytic processing occurs at critical aspartic acid residues that conform to the
caspase recognition sequence. Different caspase enzymes recognize related but different
protein sequences. Caspase-3 can be monitored using a synthetic peptide, which releases a
fluorometric moiety when cleaved by the enzyme. Prior to cleavage the substrate is nonfluorescent.
The increase in fluorescence is proportional to the amount of caspase-3 activity
present in the sample. In this assay kit, the control wells, with a precisely determined
amount of activity, are used to generate a conversion factor that allows conversion of the
nominal activity of the standards to an actual measured activity.

The protocol calls for a fluorescence measurement using a 360/40 excitation filter and a
460/40 emission filter

Plate Configuration:

Plate configuration assumes that samples will be run in duplicate. Included is a standard
curve of Caspase-6 enzyme activities (6.25 to 100 U/well). The plate includes 5 standard
concentrations, two blank wells, and two control wells. Thus 41 samples can be run on a
microplate in duplicate in addition to the standard curve.


The report is configured to provide the (1) raw fluorescence, (2) blanked fluorescence, (3)
standard curve, (4) results of the conversion factor, and (5) calculated caspase-3
concentrations of unknown samples.