Caspases,Paracaspases, and Metacaspases Methods and Protocols

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9. Syringes and 0.45 μm fi lters.


10. Ni-NTA beads (e.g., Qiagen catalog number 1018244).


11. Sonication buffer: 100 mM Tris–HCl pH 8.0, 250 mM NaCl,
    0.5 mM sucrose, EDTA-free protease inhibitors cocktail (1
    tablet/25 ml) (e.g., Roche catalog number 12483700).


12. Washing buffer 1: 100 mM Tris–HCl pH 8.0, 400 mM NaCl,
    0.5 mM sucrose, 7.5 mM β-mercaptoethanol, 0.04 % NP-40,
    EDTA-free protease inhibitors cocktail (1 tablet/25 ml).


13. Washing buffer 2: washing buffer 1 + 25 mM imidazole.


14. Elution buffer 1: 100 mM Tris–HCl pH 8.0, 250 mM NaCl,
    0.5 mM sucrose, 10 % glycerol, 7.5 mM β-mercaptoethanol,
    0.04 % NP-40, 120 mM imidazole, EDTA-free protease
    inhibitors cocktail (1 tablet/25 ml).


15. Elution buffer 2: 100 mM Tris–HCl pH 8.0, 250 mM NaCl,
    0.5 mM sucrose, 10 % glycerol, 7.5 mM β-mercaptoethanol,
    0.04 % NP-40, 250 mM imidazole, EDTA-free protease
    inhibitors cocktail (1 tablet/25 ml).


16. Elution buffer 3: 100 mM Tris–HCl pH 8.0, 250 mM NaCl,
    0.5 mM sucrose, 10 % glycerol, 7.5 mM β-mercaptoethanol,
    0.04 % NP-40, 500 mM imidazole, EDTA-free protease
    inhibitors cocktail (1 tablet/25 ml).


17. UV spectrophotometer and 96-well plate ( see Note 5 ).


18. Fluorogenic caspase substrate: Ac-Asp-Glu-Val-Asp-AMC.


19. 2× CED-3 reaction buffer: 50 mM Tris–HCl pH 8.0, 0.5 mM
    EDTA, 0.5 mM sucrose, 5 % glycerol.


20. TNT transcription and translation-coupled rabbit reticulocyte
    system (Promega catalog number L4610).


21. [^35 S]-Methionine.


22. RNase inhibitor.


23. SDS polyacrylamide gel (PAGE).


24. Fixation buffer: 50 % methanol, 10 % glacial acetic acid.


25. Gel drying buffer: 7 % methanol, 7 % glacial acetic acid, 1 %
    glycerol.


26. Phosphorimager.


27. 30 °C incubator.


28. PVDF membrane.


29. Ponceau S solution: 2 g of Ponceau S, 30 g of trichloroacetic
    acid, 30 g of sulfosalicylic acid in 100 ml of H 2 O.


30. Destaining solution: 5 % methanol, 7.5 % glacial acetic acid.

2.2 Measuring
the Activity of
Recombinant CED-3

2.3 In Vitro Cleavage
Assay Using
Substrates Labeled by
[^35 S] Methionine

2.4 Determination of
the CED-3 Cleavage
Site in the CED-3
Substrate

In Vitro CED-3 Assays