Caspases,Paracaspases, and Metacaspases Methods and Protocols

83

pan- caspase inhibitor which will interact with all active caspases and

has been used to effi ciently detect active caspase-2 following immu-

noblotting with anti-caspase-2 antibody [ 41 , 42 ] ( see Note 8 ).

1. The affi nity label can be added directly to cells in culture
   medium. In this case, approximately 1 × 10^7 cells are left
   untreated or treated with apoptotic stimuli and then incubated
   with 50 μM biotin-VAD-FMK for 2 h at 37 °C. As a negative
   control, include cells incubated in the same volume of DMSO.


2. Lyse cells in lysis buffer followed by freeze–thaw cycles in liq-
   uid nitrogen/ice-cold water as described in Subheading 3.3.2
   ( steps 1 – 5 ). Affi nity label can also be added at this point to the
   cell extract at a fi nal concentration of 50 μM. In this case,
   dilute the affi nity label to 100 μM in Dilution Buffer, add an
   equal volume to cell extract containing 100–500 μg total pro-
   tein, and incubate at room temperature for 1 h.


3. Increase the sample volume to 1 ml in lysis buffer and add
   50 μl streptavidin-coupled Sepharose. Incubate extracts with
   the Sepharose on a rotating platform, overnight at 4 °C.


4. Precipitate the bound Sepharose by centrifugation at 2,000 × g for
   3 min and wash three times with 1 ml lysis buffer ( see Note 9 ).


5. Add an equal bed volume of 2× PLB (50 μl) and boil samples
   for 5 min.


6. Resolve proteins on 15 % SDS-PAGE and transfer to PVDF
   membrane ( see Subheading 3.3.2 , steps 9 and 10 ).


7. Detect caspase-2 by immunoblotting with anti-caspase-2 rat
   monoclonal antibody (11B4) ( see Subheading 3.3.2 , steps
   11 – 13 ).

4 Notes

1. Caspase-2 activity appears to be optimal at a slightly acidic pH
   [ 44 ], so assay buffer containing 0.1 M MES, pH 6.5, is com-
   monly used. However, protein still demonstrates activity in
   buffer containing 0.1 M HEPES, pH 7.0. Caspase-2 Assay
   Buffer without DTT can be prepared in advance and stored at
   room temperature for several months, with DTT added fresh
   as required.


2. Caspase assay buffer and cell lysis buffer should contain freshly
   prepared protease inhibitor cocktail to prevent nonspecifi c
   hydrolysis of caspase substrates during protein extract prepara-
   tion. Make sure that the protease inhibitor mix does not inhibit
   caspases.


3. There are numerous commercial sources of caspase-2 antibod-
   ies; however, many available antibodies are of poor quality.
   Specifi city and affi nity of the antibody can be established using

Assays for Studying Caspase-2