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The Laboratory of Qin Chen at the University of Arizona
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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% (see Hint #1).
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 (see Hint #2).
9. Determine fluorescence at an excitation wavelength of 365 nm and an emission wave length of 450 nm.
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| Substrate Stock |
| Store at -20°C
1.6 mM Apopain/CPP-32 Substrate
in DMSO (CAUTION! see Hint #1)
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| Substrate Buffer |
| 1 part 50 mM DTT
5 parts HEPES/NaCl Stock Buffer
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| 50 mM DTT |
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| HEPES/NaCl Stock Buffer |
| 0.2 M NaCl
40 mM HEPES
pH 7.5
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| Lysis Bufer |
| 50 mM Tris
150 mM NaCl
0.5 mM EDTA
0.5% NP-40
pH 7.5
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HEPES
Sodium Chloride
DTT
DMSO
EDTA
Tris
NP-40
Sodium Chloride
Apopain/CPP-32 Substrate
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1. For example, for 10 μg of protein per reaction well add 11 μg 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.
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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.
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