Caspases,Paracaspases, and Metacaspases Methods and Protocols

164

Caspase inhibitor stock solutions: Prepare stock solutions of each

specifi c caspase inhibitor (Z-VAD-FMK, Z-XXXD-FMK, or Z-

VDVAD-FMK) to be tested by dissolving in DMSO at a concentra-

tion of 10–20 mM. Make aliquots and store at −20 °C ( see Note 15 ).

3 Methods

All procedures are performed at room temperature unless other-

wise stated.

1. Induce apoptosis in cells by desired way. Concurrently incu-
   bate a control culture without induction.


2. Collect 0.5–1 × 10^6 cells in a tube and centrifuge for 5 min at
   150 × g , 4 °C.


3. Wash cells in PBS, centrifuge again, and discard supernatant.


4. Resuspend cells in cold 50–100 μL of cold PBS. Put the sam-
   ples on ice ( see Note 16 ).


5. Determine the total protein concentration in the samples by
   removal of 1–2 μL of homogenous cell suspension for further
   analysis using the BCA protein assay ( see Note 17 ).


6. Add the appropriate volume of 5× sample buffer to a fi nal dilu-
   tion of 1×.


7. Boil samples for 5–10 min, preferentially in a high speed orbital
   shaker to eliminate the problem with high viscosity due to the
   release of chromosomal DNA ( see Note 18 ). Cool down sam-
   ples to room temperature before loading to the gel. Samples
   can be stored in −20 °C for several months ( see Note 19 ).


8. Assemble the glass plates for the Mini PROTEAN ® 3 or equiv-
   alent electrophoresis system according to the manufacturer’s
   instructions.


9. Prepare separating gel. Since the molecular masses of procas-
   pases vary between 30 and 55 kDa, and their processed (active)
   subunits detected by most antibodies are around 18 kDa, a
   15 % gel is recommended. To make one set (two gels) with
   1.0-mm-thick spacers, combine the following components:
   4.8 mL H 2 O.
   5 mL Resolving gel buffer, 1.5 M Tris–HCl, pH 8.8.
   200 μL 10 % SDS.
   10 mL Protogel.
   60 μL 10 % APS.
   33 μL TEMED.

2.6 Inhibition of
Caspase Activity in
Cell Cultures

3.1 Immunoblot
Analysis of
Procaspase
Processing

3.1.1 Sample
Preparation

3.1.2 SDS-PAGE

Magnus Olsson and Boris Zhivotovsky

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