Caspases,Paracaspases, and Metacaspases Methods and Protocols

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This protocol is almost identical to that described in the previous

section. The only difference being that this protocol utilizes a chro-

mophore-linked caspase substrate (Ac-DEVD-pNA). It must be

pointed out that this method is less sensitive and more time consum-

ing than the pro-luminescent caspase-3/7 substrate-based method.

However, this method is cheaper; the substrate is less expensive and

requires only the ability to read absorbance of a 96-well plate.

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.


3. Incubate the egg extract at room temperature for 6 h.


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 remains at room temperature
   ( see Note 14 ).


5. Add 10 μL of Ac-DEVD-pNA substrate to each well. Mix
   briefl y and gently, and then incubate the plate at 37 °C for 1 h.


6. Read the plate at 405 nm on a microplate reader.

Aside from the caspase-3/7 activity assays, there are currently no

means by which to directly measure endogenous caspase activation

in X. laevis egg extract. No commercial antibody is available at

present that recognizes Xenopus caspases. Assays similar to those

described in Subheadings 3.3 and 3.4 exist for other caspases, such

as caspase-2, caspase-8, and caspase-9; however, these assays are

not specifi c. Caspase-3 has been shown to more effi ciently cleave

most substrates than the caspases that the substrates are reportedly

specifi c for [ 19 ]. Therefore, we use the protocol described below

to quickly and simply analyze the processing of caspases other than

3 and 7 in the egg extract, caspase-2 in this case. This protocol in

vitro translates caspase-2 using a rabbit reticulocyte system

(Promega). This exogenous caspase-2 is then added to the egg

extract, and its processing is monitored over time by autoradiogra-

phy and used as a marker for caspase-2 activation with a character-

istic cleavage pattern [ 13 ]. This protocol can also be adapted for

use with any other caspase; all that is needed is plasmid DNA in a

vector that is responsive to the appropriate promoter (T7 or SP6 in

the case of the rabbit reticulocyte system).

1. Prepare the following reaction, using the components of the
   TNT-coupled reticulocyte lysate system (Promega), in a
   RNase/DNase-free Eppendorf:
   (a) 25 μL rabbit reticulocyte lysate.
   (b) 2 μL TNT buffer.

3.4 Assessment
of Caspase-3/7
Activity in X. laevis
Egg Extract Using a
Chromophore- Linked
Caspase Substrate

3.5 Assessment of
Caspase-2 Processing
in X. laevis Egg Extract
Using a^35 S - Labeled In
Vitro-Translated
Protein

Methods for the Study of Caspase Activation in the Xenopus laevis Oocyte...