Caspases,Paracaspases, and Metacaspases Methods and Protocols

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protocol utilizes a pro-luminescent caspase-3/7 substrate

(Promega) containing the DEVD motif. In the presence of active

caspase-3/7, this substrate will be cleaved and release aminolucif-

erin, a substrate of luciferase that will result in the production of

light. When this substrate is incubated in the egg extract undergo-

ing apoptosis (and active caspase-3 and/or caspase-7), the sub-

strate is cleaved, and a luminescent signal is produced proportional

to the level of caspase- 3/7 activity.

1. Pipette 85 μL of DEVDase buffer per well of a 96-well plate
   for each sample and/or time point to be assayed.


2. Supplement egg extract from Subheading 3.1 with 1:20 dilu-
   tion of 20× EM. Add any treatments that you wish to examine
   ( see Note 13 ).


3. Incubate the egg extract at room temperature for 6 h. A good
   quality extract will undergo caspase activation at approximately
   3 or 4 h ( see Fig. 2 ).


4. Add 3 μL of egg extract per well for each time point and/or
   sample. The plate should be kept on ice while samples are
   being taken, while the extract should remain at room tempera-
   ture ( see Note 14 ).


5. Add 5 μL of the Caspase-Glo 3/7 assay substrate (Promega)
   to each well. Mix briefl y and gently, and then incubate the
   plate at 37 °C for 15 min.


6. Analyze the luminescent output of the plate using 3 s inte-
   grated readings on a microplate reader.

Fig. 2 Spontaneous apoptosis in X. laevis egg extracts. Egg extract was incu-

bated at room temperature, and aliquots were taken at indicated time points and

analyzed for caspase-3/7 activity as described in Subheading 3.3. Note that

addition of G6P to a separate aliquot of extract inhibited this spontaneous apop-

tosis (McCoy and Nutt, unpublished data)

Francis McCoy et al.

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