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- It is best to prepare a “stock” egg extract supplemented with
EM and then divide this between each sample you wish to assay. - The temperature in our laboratory is typically around
18–21 °C. - Take care not to run the gel too far as one of the cleavage
products of caspase-2 is low molecular weight and can easily
be lost off the bottom of the gel. - The length of exposure will depend on the age of the [^35 S]
methionine. With fresh [^35 S]methionine, typically an over-
night or one day exposure should be enough to give a reason-
ably strong signal. - An aliquot of extract could also be taken at each time point
and used to assess caspase-3/7 activity using the assay described
in Subheading 3.3. - We recommend cutting the membrane at or below ~75 kDa
mark, as the antibody recognizes a nonspecifi c protein that can
reduce overall signal intensity of the bands of interest. - Apoptotic nuclei should appear condensed and fragmented.
The difference with early time point nuclei, or nuclei in extracts
where apoptosis has been inhibited, should be very apparent. - This method will only summarize the procedure for physical
removal of oocytes from female frogs. A more detailed descrip-
tion of Xenopus anatomy and dissection procedure can be
found in Ref. [ 21 ]. - When digestion is complete, the oocytes will be detached from
each other and any connective tissue. - The oocytes should be sorted the same day they are harvested
from the frog and stored for no more than one night at 18 °C,
and the experiment performed the next day. It is also a good
idea to quickly sort the oocytes again the next morning to
remove any that died overnight. - The Ficoll will need to be dissolved in dH 2 O fi rst, by briefl y
microwaving until dissolved. This needs to be done prior to
adding the MMR solution, as the heating will destroy the
EDTA in MMR. - The settings we use on our micropipette puller are as follows:
P = 500, heat = 350, pull = 200, V = 100, and time = 150. These
needles then need to be cut, beveled, and calibrated to the
point where a single injection will inject the desired volume
(10—25 nL). - Care must be taken when physically handling the oocytes, as
they are easily damaged when too much force is applied. We
use a plastic Pasteur pipette, with the end cut off to create a
wider opening for the oocytes, preventing them from becom-
ing damaged.
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