Caspases,Paracaspases, and Metacaspases Methods and Protocols

160

Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-fl uoromethylk-

etone) is a pan-caspase inhibitor that irreversibly binds to the cata-

lytic site of caspases thereby preventing their activity, further

processing and apoptosis. Similar to synthetic substrates used for

the detection of caspase activities, tetrapeptides and pentapeptides

designed to block the catalytic site of individual caspases are also

commercially available. However, as previously discussed in the

section describing the caspase activity assay, the use of synthetic

amino acid target motifs to defi ne individual caspases and isolated

apoptotic signaling pathways is debated [ 13 ]. Since the peptides

described bind to the catalytic site of active caspases they may also

be used as markers. Exposure of live cells to fl uorochrome-labeled

inhibitors of caspases (FLICAs) results in uptake of these reagents

by apoptotic cells. After fi xation with formaldehyde, cells labeled

with FLICAs can be examined by fl uorescence microscopy, or

quantifi ed by fl ow cytometry [ 15 ].

2 Materials

All solutions should be prepared in ultrapure water (18.3 MΩ at

25 °C) and by using analytical grade chemicals. Unless indicated

otherwise, all solutions can be prepared and stored at room

temperature.

1. Phosphate-buffered saline (PBS), pH 7.4.


2. Pierce™ BCA Protein Assay Kit (Thermo Scientifi c) or similar.


3. 5× Sample (Laemmli) buffer: 625 μL 1 M Tris–HCl, pH 6.8,
   1 mL glycerol, 2 mL 10 % SDS, 0.5 mL 0.5 % (w/v) bromo-
   phenol blue in water, 0.5 mL 2-mercaptoethanol. Add water
   to 10 mL, aliquot and store at −20 °C ( see Note 1 ).


4. Mini PROTEAN ® 3 electrophoresis system or equivalent
   equipment.


5. Prot/Elec Tips (Bio-Rad) or similar tips.


6. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Weigh 181.7 g
   Tris and dissolve in 800 mL water. Titrate the solution of Tris
   with hydrochloric acid (HCl) until the correct pH is reached
   ( see Notes 2 and 3 ). Make up to 1 L with water. Store at 4 °C.


7. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Weigh 60.6 g Tris
   and prepare a 1 L solution as in previous step. Store at 4 °C.


8. Protogel, 30 % (w/v) Acrylamide: 0.8 % (w/v) Bis-Acrylamide
   Stock solution (37.5:1).


9. N , N , N ′, N ′-tetramethylethylenediamine 99 % (TEMED).


10. Ammonium persulfate (APS): 10 % solution in water
    ( see Note 4 ).

2.1 Immunoblot
Analysis of
Procaspase
Processing

2.1.1 Sample
Preparation

2.1.2 SDS-PAGE

Magnus Olsson and Boris Zhivotovsky

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