Caspases,Paracaspases, and Metacaspases Methods and Protocols

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Exogenous sperm nuclei can even be added to X. laevis egg extract,

and nuclear morphology can be studied as a technique for measur-

ing apoptosis (Subheading 3.9 ). Caspase activation can also be

measured by analyzing cleavage of caspase substrates, e.g., PARP,

in the extract by measuring PARP cleavage (Subheading 3.8 ).

2 Materials

1. Mature female frogs (e.g., Nasco; http://www.enasco.com/
   page/xenopus ).


2. Frog aquarium.


3. Pregnant mare serum gonadotropin (PMSG, Calbiochem).
   Reconstituted in autoclaved, sterile fi ltered (0.22 μm fi lter)
   ddH 2 O, to a stock concentration of 200 U/mL. Store at
   −20 °C.


4. Human chorionic gonadotropin (hCG, Sigma-Aldrich).
   Reconstituted in autoclaved, sterile fi ltered (0.22 μm fi lter)
   ddH 2 O, to a stock concentration of 1,000 U/mL. Store at
   4 °C.


5. 2 % L-cysteine (w/v), pH 8.0.


6. 40× Marc’s Modifi ed Ringer’s solution (MMR): 50 mM
   HEPES pH 7.7, 1 M NaCl, 20 mM KCl, 10 mM MgSO 4 7H 2 O,
   25 mM CaCl 2 , and 2 mM EDTA.


7. 10× egg lysis buffer (ELB): 100 mM HEPES pH 7.7, 25 mM
   MgCl 2 , and 500 mM KCl.


8. Cycloheximide. Reconstituted in ddH 2 O to a stock concentra-
   tion of 10 mg/mL.


9. Aprotinin. Reconstituted in ddH 2 O to a stock concentration
   of 5 mg/mL.


10. Leupeptin. Reconstituted in ddH 2 O to a stock concentration
    of 5 mg/mL.


11. Cytochalasin B. Reconstituted in dimethyl sulfoxide (DMSO)
    to a stock concentration of 5 mg/mL.


12. 200-mL glass beaker.


13. 14-mL polypropylene round-bottom tube.


14. IEC clinical centrifuge (or equivalent).


15. Avanti J-26 XPI centrifuge (Beckman Coulter), with a JS 13.1
    swinging bucket rotor (or equivalent).


16. 10-mL syringe.


17. 16-gauge needle.


18. 15-mL polypropylene conical tubes.

2.1 Collection
of X. laevis Eggs
and Preparation
of Interphase Egg
Extract

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