Caspases,Paracaspases, and Metacaspases Methods and Protocols

122

1. X. laevis egg extract.


2. 2.5-mL (11 × 34 mm) polycarbonate centrifuge tubes (e.g.,
   Beckman).


3. TLS ultracentrifuge and TLS-55 swinging bucket rotor
   (Beckman) or equivalent.


4. X. laevis egg extract.


5. 20× energy mix (EM): 7.5 mM Phosphocreatine, 1 mM ATP,
   0.1 mM EGTA, and 1 mM MgCl 2.


6. White 96-well, fl at-bottomed plate.


7. DEVDase buffer: 50 mM HEPES pH 7.5, 100 mM NaCl,
   0.1 % CHAPS, 10 mM DTT, 1 mM EDTA, and 10 % glycerol.


8. Caspase-Glo 3/7 assay substrate (Promega).


9. SpectraMax M3 microplate reader (Molecular Devices) or
   equivalent.


10. X. laevis egg extract.


11. 20× energy mix (EM): 7.5 mM Phosphocreatine, 1 mM ATP,
    0.1 mM EGTA, and 1 mM MgCl 2.


12. Clear 96-well, fl at-bottomed plate.


13. DEVDase buffer: 50 mM HEPES pH 7.5, 100 mM NaCl,
    0.1 % CHAPS, 10 mM DTT, 1 mM EDTA, and 10 % glycerol.


14. Ac-DEVD-pNA (e.g., from Enzo Life Sciences).


15. SpectraMax M3 microplate reader (Molecular Devices) or
    equivalent.


16. X. laevis caspase-2 DNA (pSP64T).


17. TNT-coupled reticulocyte lysate system (Promega).
    (a) Rabbit reticulocyte lysate.
    (b) Reaction buffer.
    (c) SP6 RNA polymerase.
    (d) Amino acid mixture (minus methionine).
    (e) [^35 S]methionine.
    (f) RNasin ribonuclease inhibitor.


18. Nuclease free water.


19. X. laevis egg extract.


20. SDS sample buffer: 150 mM Tris–HCl pH 6.8, 10 % SDS ,
    30 % Glycerol, and 5 % β-mercaptoethanol.


21. SDS-PAGE apparatus.


22. Gel dryer.


23. X-ray fi lm and cassette.

2.2 Fractionation
of Egg Cytosols

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

2.4 Assessment
of Caspase-3/7
Activity in X. laevis
Egg Extract Using a
Chromophore- Linked
Caspase Substrate

2.5 Assessment
of Caspase-2
Processing in X. laevis
Egg Extract Using
a^35 S - Labeled In
Vitro-Translated
Protein

Francis McCoy et al.

http://www.ebook3000.com