Caspases,Paracaspases, and Metacaspases Methods and Protocols

127

1. Transfer the crude egg extract prepared as described in the
   previous section to a 2.5-mL polycarbonate centrifuge tube.
   All the steps in this procedure should be performed on ice
   ( see Note 10 ).


2. Centrifuge the crude extract at 200,000 × g for 70 min at 4 °C.


3. Following this spin the extract will separate into a clear cyto-
   plasmic layer, with two membrane layers below: a pale, light
   membrane fraction and a dark, heavy membrane fraction
   directly below this. There will be a fi nal very dark layer at the
   bottom of the tube, consisting of mainly glycogen, ribosomes,
   etc. (Fig. 1c ) ( see Note 11 ).


4. Carefully remove the clear cytoplasmic layer, taking care not
   to disturb the underlying membrane layers, and transfer it to a
   fresh 2.5-mL centrifuge tube.


5. Centrifuge the cytoplasmic fraction again at 200,000 × g for
   25 min. This step removes any remaining membrane
   contaminants.


6. Remove the clear cytoplasmic fraction, aliquot it, and store at
   −80 °C until ready to use.

Activation of caspases, responsible for the ordered dismantling of

the cell, lies at the apex of the apoptotic program. Apoptosis gener-

ally culminates in the activation of the executioner caspase-3 and

caspase-7 ( see Note 12 ). These caspases have a very defi ned and

specifi c cleavage motif, cleaving after the second aspartic acid resi-

due in the DEVD motif. This specifi city allows for the activation of

caspase-3/7 to be studied very easily in vitro. The following

3.3 Assessment
of Caspase-3/7
Activity in X. laevis
Egg Extract Using
a Luminescent
Caspase Substrate

Fig. 1 Collection of X. laevis eggs and preparation of interphase egg extract and fraction of egg extract cytosol.
( a ) Eggs are packed by low-speed centrifugation and excess buffer is removed. Following the addition of
cycloheximide, cytochalasin B, aprotinin, and leupeptin, the eggs are lysed by centrifugation at 15,600 × g for
10 min. ( b ) The eggs are fractionated into distinct layers following high-speed centrifugation: a lipid layer on
top; a pellet of yolk proteins, glycogen, pigment granules, and nuclei; and a middle layer of crude egg extract.
( c ) Fractionation of crude egg extract is achieved by ultracentrifugation at 200,000 × g. This separates the
crude extract into a clear cytosolic fraction on top, a pale light membrane fraction just below, a darker heavy
membrane fraction, and a pellet consisting mainly of glycogen and ribosomes

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