130
(c) 1 μL SP6 polymerase.
(d) 1 μL amino acid mixture, minus methionine (20 μM fi nal
concentration).
(e) 2 μL [^35 S]methionine (20 μCi).
(f) 1 μL RNasin (40 μM fi nal concentration ).
(g) 2 μL pSP64T caspase-2 DNA (0.4 μg fi nal concentration).
(h) 16 μL nuclease free water, to a fi nal volume of 50 μL.- Incubate this reaction at 30 °C for 2 h.
- Aliquot and store at −20 °C if not using immediately.
- Add 8 μL of this TNT reaction to 50 μL of egg extract from
Subheading 3.1 supplemented with EM, and incubate at room
temperature for ~6 h. For each time point (either every hour
or every 2 h), take 5 μL of this reaction and add it to 30 μL of
SDS sample buffer. - Heat these samples for 3 min at 80 °C.
- Analyze these samples by SDS-PAGE, loading 15 μL of sam-
ple/well ( see Note 15 ). - Dry the gel using an appropriate gel-drying apparatus (we use
a model 583 gel dryer from Bio-Rad), and expose to X-ray
fi lm ( see Note 16 ).
The X. laevis egg extract recapitulates all the key hallmarks of apop-
tosis, including cytochrome c release. Cytochrome c release, which
follows mitochondrial outer membrane permeabilization (MOMP),
is a key event in the intrinsic apoptotic pathway. Once released into
the cytosol, cytochrome c forms the apoptosome; a caspase-9-acti-
vating platform composed of cytochrome c , APAF-1, dATP, and
procaspase-9. Monitoring release of cytochrome c is a very useful
tool in dissecting the apoptotic cascade under varying treatment
conditions. The fi rst protocol presented below (Subheading 3.6.1 )
describes how to very simply, and quickly, monitor cytochrome c
release by removing mitochondria via fi ltration and subsequently
immunoblotting for cytochrome c in the cytosolic fraction.
The X. laevis egg extract requires the presence of mitochondria
to initiate apoptosis and caspase activation [ 1 ]. Subheading 3.2
described how to isolate the cytosolic fraction of egg extract, frac-
tionating heavy and light membranes. Lacking mitochondria, this
cytosolic fraction will not initiate apoptosis; however, it is still com-
petent to undergo apoptosis, utilizing the post-mitochondrial
machinery (caspase-9, apoptosome, etc.). The second protocol
below (Subheading 3.6.2 ) describes how to initiate caspase activa-
tion in this cytosolic fraction, by addition of exogenous cytochrome
c and utilizing the protocol described in Subheading 3.3. This
cytosolic fraction can provide a simplifi ed, tractable system in
which to study the mechanisms of caspase activation under many
different settings.3.6 Assessment
of Cytochrome
c Release from
Mitochondria
in X. laevis Egg
Extract, and Induction
of Apoptosis in
Cytosolic Fractions
of Egg Extract Using
Cytochrome c
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