137- By lysing the eggs in the presence of EGTA, the effect of cal-
cium will be blocked, and the eggs will remain arrested in
metaphase of meiosis II. This extract is very resistant to induc-
tion of apoptosis [ 15 ]. - X. laevis eggs are surrounded by a transparent jelly coat, which
is removed by cysteine. Leaving the eggs in cysteine for too
long can cause the eggs to lyse. When the eggs pack closely
together, this is a sign that they are de-jellied. This will usually
occur before the 5 min period is complete. - Once the jelly coat is removed, the following steps need to be
done without pause. The eggs are more vulnerable without
their jelly coat and taking too long can decrease the overall
quality of the fi nal extract. - This method may leave behind some bad eggs; however, the
number of bad eggs is typically very small and not enough to
affect the functionality of the fi nal extract. If you require all
bad eggs to be removed, an alternative method is to pour the
eggs into a petri dish and remove the bad eggs with a Pasteur
pipette. The pipette should be cut to provide a wider tip, so as
to avoid lysing the eggs. - Aprotinin and leupeptin are protease inhibitors; aprotinin
inhibits trypsin-, chymotrypsin-, and plasmin-like proteases,
while leupeptin inhibits lysosomal proteases. Cytochalasin B
inhibits actin polymerization and cycloheximide inhibits pro-
tein synthesis. - Be sure not to puncture too close to the dark bottom layer,
and ensure the needle bevel is pointing up at fi rst. As the lipid
layer moves closer, slowly turn the bevel 90° so that it is facing
away from the lipid layer. It is important to not take up any of
the lipid layer or bottom pigmented layer. We typically do not
attempt to remove all of the crude extract as this will more
likely lead to contamination. Instead, by slowly and carefully
removing all but a small portion of the crude extract layer, we
avoid any contamination by the other layers. It is also common
for the needle to be clogged (by the plastic of the tube during
penetration) resulting in extremely slow removal of the extract.
When this occurs, we insert a second needle perpendicular to
the fi rst needle and repeat the process. Do not remove the fi rst
needle as this will lead to loss of extract. - Be sure to fi ll the tube at least ¾ full to avoid the tube collaps-
ing during centrifugation. - There may also be a thin pale/yellow layer of lipid above the
clear cytoplasmic layer. This is normal and can be easily
removed or avoided. - There are also caspase-independent pathways of apoptosis;
however, they will not be discussed here.
Methods for the Study of Caspase Activation in the Xenopus laevis Oocyte...