Caspases,Paracaspases, and Metacaspases Methods and Protocols

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1. Supplement egg extract from Subheading 3.1 with 1:20 dilu-
   tion of 20× EM. Divide this extract into two separate aliquots,
   one for analyzing cytochrome c release and one for analyzing
   caspase-2 processing ( see Note 17 ).


2. Add 8 μL of the TNT reaction from Subheading 3.5 per 50 μL
   of egg extract from Subheading 3.1 for analysis of caspase-2
   processing.


3. Divide these two sets of samples (one for cytochrome c release
   and one for caspase-2 processing) into two separate aliquots.
   Supplement one of these aliquots with 500 nM recombinant
   BCL-xL, and leave the other untreated to serve as control.


4. Incubate the samples at room temperature for ~6 h. Every
   30–60 min, take 20 μL to perform the cytochrome c release
   assay from Subheading 3.6 , and every 1–2 h take 5 μL of the
   extract supplemented with TNT caspase-2 and add it to 30 μL
   of SDS sample buffer and process as described in
   Subheading 3.5.

Poly(ADP-ribose) polymerase (PARP) is a family of proteins that

play an important role in DNA repair. PARP detects single-strand

breaks in DNA, binds to the DNA, and initiates the repair process.

PARP is a 116 kDa protein that during apoptosis is cleaved by

caspase- 3/7 thus preventing it from initiating DNA repair. This

cleavage event can be detected very simply by immunoblotting and

has been used extensively in mammalian systems to detect induc-

tion of apoptosis. We show here that it can also be used in X. laevis

egg extracts as a robust marker for induction of apoptosis and cas-

pase activation.

1. Supplement egg extract from Subheading 3.1 with 1:20 dilu-
   tion of 20× EM.


2. Incubate this egg extract at room temperature for 6 h. Every
   hour, take a 4 μL aliquot of extract and add to 30 μL of SDS
   sample buffer.


3. Subject 10 μL of each sample to SDS-PAGE gel (using a
   4–20 % gradient gel).


4. Immunoblot with an anti-PARP antibody. A 1:1,000 primary
   antibody concentration and a 1:2,000 secondary antibody
   concentration are recommended ( see Note 18 ). PARP has a
   very distinctive cleavage pattern, with full-length PARP at
   ~116 kDa and a cleavage product at ~86 kDa (Fig. 3 ).

Another key hallmark of apoptosis and caspase activation is nuclear

fragmentation and morphology. Although X. laevis egg extracts

lack nuclei, addition of exogenous sperm chromatin results in for-

mation of synthetic nuclei that appear condensed and fragmented

in apoptotic extracts. This protocol has been used since the

3.8 Assessment
of PARP Cleavage
by Western Blot

3.9 Assessment
of Nuclear Morphology
in X. laevis Egg
Extracts

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