Enzyme Evaluation Protocol
This protocol is used to measure caspase enzyme activity. The synthetic fluorogenic peptide Ac-IETD-AMC (Prod. No. ALX-260-042) is used as the caspase enzyme substrate. The enzyme cleaves the substrate releasing the fluorescent AMC (7-amino-4-methylcoumarin). AMC release can be measured by spectrofluorometry using UV.

1. Add 20mM of Ac-IETD-AMC (Prod. No. ALX-260-042) to 1ml of assay buffer (20mM PIPES, 100mM sodium chloride, 10mM DTT, 1mM EDTA, 0.1% (w/v) CHAPS, 10% sucrose, pH 7.2). Add the appropriate amount of  active caspase to the mixture (50ng/ml).
2. Incubate for 1 hour at 37°C.
3. Measure the AMC liberated from the Ac-IETD-AMC using a spectrofluorometer with an excitation wavelength of 380nm and an emission wavelength of 440nm.

Note: Enzyme activity can also be measured using the caspase enzyme substrate Ac-IETD-pNA (Prod. No. ALX-260-045) at 37°C in a spectromax at 405nm wavelength.

Swiss-Prot link Q92851: Caspase-10 (human) (precursor)
AfCS Signalling Gateway link A000493: Caspase-10 (human)