Please find the following protocol:

CPP32 Activity Assay Using CPP32 Fluorogenic Substrate


Procedure:

1. Determine the protein concentration of the sample to be analyzed (see protocol on Quantification of Protein Samples).

For each reaction (prepared in triplicate for each sample):

2. To small glass vials, add between 10 to 30 μg of protein per reaction well plus 10%. For example, for 10 μg of protein per reaction well add 11 μg to the reaction well.

3. Add Lysis Buffer to a final volume of 445.5 μl.

4. Add 198 μl of Substrate Buffer.

5. Add 16.5 μl of 1600 μM Substrate Stock and vortex.

6. The final reaction volume should be 660 μl.

7. Incubate at 37°C for 1 hour.

8. Pipette 200 μl from each reaction volume and distribute into a 96-well plate. Each reaction should be analyzed in triplicate. Thus each sample is reacted in triplicate and each reaction is analyzed in triplicate.

9. Determine fluorescence at an excitation wavelength of 365 nm and an emission wave length of 450 nm.


Solutions:

Substrate Buffer
1 part 50 mM DTT
5 parts HEPES/NaCl Stock Buffer

Substrate Stock
Store at -20°C
1.6 mM Apopain/CPP-32 Substrate in DMSO
(CAUTION Biohazard!)

50 mM DTT

HEPES/NaCl Stock Buffer
0.2 M NaCl
40 mM HEPES
pH 7.5

Lysis Bufer
50 mM Tris
150 mM NaCl
0.5 mM EDTA
0.5% NP-40
pH 7.5



Bioreagents and Chemicals:

Tris
NP-40
Sodium Chloride
Apopain/CPP-32 Substrate
HEPES
Sodium Chloride
DTT
DMSO
EDTA


References and Links:

1. Mizushima N, Koike R, Kohsaka H, Kushi Y, Handa S, Yagita H, Miyasaka N. Ceramide induces apoptosis via CPP32 activation. FEBS Lett. 1996 Oct 21;395(2-3):267-71.

http://www.bio.com/protocolstools/protocol.jhtml?id=p1990