Caspases,Paracaspases, and Metacaspases Methods and Protocols

81

coding regions containing the putative caspase-2 cleavage site

can be used [ 43 ].

2. In vitro transcription/translation of each substrate is carried
   out using the Promega TNT-Rabbit Reticulocyte Translation
   System according to the instructions provided by the manufac-
   turer. Reactions will contain 25 μl TNT lysate, 2 μl TNT reac-
   tion buffer, 1 μl RNA polymerase (T3, T7, or SP6), 1 μl amino
   acid mixture lacking methionine, 3 μl^35 S-methionine, 1 μl
   ribonuclease inhibitor, 1 μg plasmid DNA, and sterile RNase-
   free water to a fi nal volume of 50 μl.


3. Incubate reactions at 30 °C for approximately 2 h.


4. Centrifuge samples at 10,000 × g for 5 min and transfer super-
   natant to a fresh tube. At this stage, the in vitro-translated
   35 S-labeled protein can be stored at −70 °C for up to 2 weeks.


5. For cleavage assays, incubate 5 μl of^35 S-labeled protein at
   37 °C for 2 h with 50–100 nM recombinant caspase-2 in cas-
   pase assay buffer in a total volume 50 μl.


6. To controls, preincubate recombinant caspase-2 with a caspase
   inhibitor (e.g., 50 μM Ac-VDVAD-CHO, z-VAD-FMK, or
   Q-VD-OPh) for 30 min prior to the addition of in vitro-
   translated^35 S-labeled protein substrate.


7. To stop reactions, add 50 μl of 2× PLB to each tube, boil for
   5 min, and centrifuge at 10,000 × g for 5 min.


8. Check substrate cleavage products by resolving^35 S-labeled
   proteins by electrophoresis on a 10–15 % polyacrylamide/SDS
   gel.


9. Once resolved, transfer proteins to PVDF membranes using
   either wet or semidry transfer apparatus (for approximately
   90 min at 130 mA).


10. Visualize^35 S-labeled protein bands by autoradiography, follow-
    ing overnight exposure to X-ray fi lm or phosphor screen.
    Longer exposure time may be required if signal intensity is low.
    An example of autoradiograph showing caspase-2 cleavage by
    recombinant caspase-2 or apoptotic lysate is shown in Fig. 2.

An alternative method to demonstrate caspase-2-mediated cleav-

age of substrate is to incubate recombinant caspase-2 with cell

extracts and observe protein substrate cleavage by immunoblotting

using specifi c antibodies against a known endogenous caspase-2

substrates.

1. To prepare protein cell extracts, cultured cells are harvested by
   gentle scraping into medium or by treatment with trypsin for
   5 min at 37 °C (for adherent cells), or cell culture medium is
   simply collected (for suspension cells). For tissues, homoge-
   nize frozen tissue in cell extraction buffer using a tissue
   homogenizer.

3.3.2 Cleavage of
Substrates in Cells Extracts
by Recombinant
Caspase-2

Assays for Studying Caspase-2