Monday, October 27, 2003

Description
CPP32 Activity Assay Using CPP32 Fluorogenic Substrate

Procedure
. 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 g of protein per reaction well plus 10% (see Hint #1).

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

4. Add 198 l of Substrate Buffer.

5. Add 16.5 l of 1600 M Substrate Stock and vortex.

6. The final reaction volume should be 660 l.

7. Incubate at 37C for 1 hour.

8. Pipette 200 l from each reaction volume and distribute into a 96-well plate (see Hint #2).

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


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


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


Substrate Stock Store at -20C
1.6 mM Apopain/CPP-32 Substrate
in DMSO (CAUTION! see Hint #1)


50 mM DTT

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



Supplies


Tips
1. For example, for 10 g of protein per reaction well add 11 g to the reaction well).

2. Each reaction should be analyzed in triplicate. Thus each sample is reacted in triplicate and each reaction is analyzed in triplicate.