Caspases,Paracaspases, and Metacaspases Methods and Protocols

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1. X. laevis egg extract.


2. 20× EM.


3. Centrifuge fi lter, PVDF 0.1 μm (Millipore, cat. # UFC30VVNB
   or similar).


4. SDS sample buffer.


5. SDS-PAGE and Western blot apparatus.


6. Cytochrome c antibody (BD Pharmingen, cat # 556432 or
   similar).


7. X. laevis egg extract cytosolic fraction (from Subheading 3.2 ).


8. 20× EM.


9. Materials from Subheading 2.3 to analyze caspase-3/7 activity.


10. Recombinant cytochrome c ( Equine , Sigma-Aldrich or similar).


11. SDS sample buffer.


12. SDS-PAGE and Western blot apparatus.


13. X. laevis egg extract.


14. 20× EM.


15. Recombinant mouse tBID (ProSpec, cat # PRO-644 or
    similar).


16. Recombinant human BCL-xL (Sigma-Aldrich, cat # SRP0187
    or similar).


17. Glucose 6-phosphate sodium salt.


18. Materials from Subheading 2.3 to analyze caspase-3/7 activity.


19. Materials from Subheading 2.6 to analyze cytochrome c
    release.


20. Materials from Subheading 2.5 to analyze caspase-2 processing.


21. X. laevis egg extract.


22. 20× EM.


23. SDS sample buffer.


24. SDS-PAGE and Western blot apparatus.


25. PARP antibody (Novus Bio cat #NB001-64828 or similar).


26. X. laevis egg extract.


27. 20× EM.


28. Sperm chromatin. See Deming and Kornbluth for protocol [ 18 ].


29. Hoechst stain.


30. Microscope slide and cover slip.


31. Fluorescent microscope.

2.5.1 Assessment
of Cytochrome c Release
from Mitochondria
in X. laevis Egg Extract

2.5.2 Induction
of Apoptosis in Cytosolic
Fractions of X. laevis
Egg Extract Using
Cytochrome c

2.6 Using
Recombinant tBID
and BCL-xL to
Determine if Inhibition
of Caspase Activation
Lies Upstream of
Mitochondria and
BCL-2 Family Proteins

2.7 Assessment
of PARP Cleavage
by Western Blot

2.8 Assessment
of Nuclear Morphology
in X. laevis Egg
Extracts

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