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- Wash eggs three times in 1× ELB. At this stage, bad or poor
quality eggs can be removed. Mature, good quality, X. laevis
eggs have a very distinct appearance. They have a dark half
(animal pole) on top and a light half (vegetable pole) on bot-
tom. Bad eggs vary in appearance, but any eggs that are large,
puffy, white, and/or not uniformly pigmented with a distinct
dark and light half should be removed. We remove bad eggs
by essentially pouring off as many bad eggs as possible. The
bad eggs are less dense than the good eggs, and so due to the
sucrose in ELB these bad eggs will fl oat closer to the top of
the beaker when the ELB is fi rst poured in, allowing them to
be poured off while retaining the good eggs ( see Note 7 ).
Lyse eggs - Pour the eggs into a 14-mL polypropylene round-bottom
tube. From this point on, the eggs should be kept on ice as
much as possible. - Centrifuge the eggs in a clinical centrifuge at approximately
1,500 × g for 25–30 s and remove any excess buffer from the
packed eggs. - Add the following inhibitors from stocks based on the volume
of packed eggs ( see Note 8 ):
(a) 5 μg/mL aprotinin—1 μL/mL of packed eggs
(b) 5 μg/mL leupeptin—1 μL/mL of packed eggs
(c) 5 μg/mL cytochalasin B—1 μL/mL of packed eggs
(d) 50 μg/mL cycloheximide—5 μL/mL of packed eggs - Lyse the eggs by centrifugation at 15,600 × g for 10 min at
4 °C. We use an Avanti J-26 XPI centrifuge (Beckman
Coulter), with a JS 13.1 swinging bucket rotor. - Following centrifugation, the eggs will separate into three dis-
tinct layers: a yellow layer on top that consists of lipids, the
crude egg extract layer in the middle (this can vary in color
from a pale to dark brown color), and a dark bottom layer con-
sisting mainly of yolk proteins, glycogen, and pigment gran-
ules. The nuclei will also be in this bottom layer ( see Fig. 1a, b ). - Remove the crude egg extract (the middle layer) using a 16-G
needle and 10-mL syringe. Firmly yet carefully pierce the side
of the tube, taking care not to puncture the opposite side of the
tube. Slowly draw out the crude extract ( see Note 9 ). Transfer
the extract to a 15-mL polypropylene conical tube on ice.
Using the following protocol, it will be possible to separate the
cytosolic fraction from crude extract. This cytosolic fraction will be
competent to undergo apoptosis with the addition of cytochrome
c as described later (Subheading 3.6.2 ).3.2 Fractionation
of Egg Cytosols
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