This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

CASPASES
Ann Zeuner, Maria Rita Rippo, Ruggero De Maria
and Roberto Testi
 
Dept. Experimental Medicine and Biochemical Sciences,
University of Rome Torvergata, Rome, Italy
EMail: tesrob@flashnet.it



1. Introduction
  2A. First protocol: cleavage of synthetic substrates 2A.1. Materials  2A.2. Methodology

Induce apoptosis in cells by the desired method. To evaluate protease activity in terms of fold-increase, concurrently incubate a negative control (control #1) without induction.

  1. Count cells and pellet 106 cells for each sample.
  2. Wash cells once with cold PBS.
  3. Resuspend cells in 50 µl of chilled lysis buffer and incubate on ice for 20 min.
  4. Lyse the cells by 3-4 cycles of freezing and thawing.
  5. Pellet insoluble material by centrifugation for 15 min at 14,000 rpm at 4°C. The clear supernatant can be used immediately or stored at -70°C for assay at a later time.
  6. Optional: to verify that the signal detected is attributable to CPP32 protease activity, and to assess the fluorescence contribution of the subpopulation of apoptotic cells normally present in culture, a negative control #2 can be performed by treating an induced sample with the CPP32 inhibitor DEVD-CHO before incubation with the fluorescent substrate.
  7. To do so, add 50 µl of 2x Reaction Buffer and 1 ml of CPP32 inhibitor (from 1 mM solution) to an induced sample lysate and incubate at 30°C for 30 min (if your are measuring ICE activity, do the same with YVAD-CHO). Keep the other lysates on ice during this time. Then proceed to step 9, adding reaction buffer to the samples that were held on ice, and continue.
  8. Add 50 µl of 2x Reaction Buffer to each reaction.
  9. Add to each tube 5 µl of CPP32 substrate (DEVD-AFC) or ICE substrate (YVAD-AFC) from 1 mM stock solution to make 50 µM final concentration. Incubate at 37°C for 1 hr.
  10. Read the samples in a fluorometer equipped with a 400 nm excitation filter and a 505 nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. The typical fluorescence emission of apoptotic samples is 4-7 fold above that of the uninduced control sample.
  11. NOTE: As an alternative to DEVD-AFC, it is possible to use the fluorogenic substrates Ac-DEVD-AMC (7-amino-4- methylcoumarin) or Ac-YVAD-AMC, both available from Bachem, Bubendorf, Switzerland. In this case it will be necessary to read the samples using an excitation wavelength of 360 nm and an emission wavelength of 480 nm.

   

3A. Commentary

 
3A.1. Troubleshooting

2B. Second protocol: PARP cleavage assay
  2B.1. Materials 2B.2. Methodology
 
    1. Perform PARP transcription and translation using the TNT kit according to the manufacturer's recommendations. Usually 5 m l of the 50 m l final reaction mixture contain a sufficient amount of 35S-labeled PARP.
    2. Induce apoptosis in cells by the desired method, keeping an uninduced sample for negative control.
    3. Collect cells (0.5-3x106/sample), wash once with cold PBS and resuspend in 50 m l chilled Cell Lysis Buffer.
    4. Keep 20 min on ice.
    5. Proceed with lysis by 3-4 cycles of freezing and thawing.
    6. Pellet insoluble material by centrifugation for 15 min at 14,000 rpm at 4°C.
    7. After centrifugation, collect the supernatants and determine protein concentration of 2-5 m l, while keeping the rest of the lysates on ice. Remaining lysates can be frozen at -70°C for assay at a later time.
    8. In a 0.5 ml reaction tube kept on ice, mix together 10 µg cell lysate, 4 µl 5x Reaction Buffer, 5 µl 35S-labeled PARP, and bring the final volume to 20 µl with H2O.
    9. Incubate 1 hr at 37°C.
    10. Stop the reaction by adding 5 µl of 5x Laemmli Sample Buffer.
    11. Boil the samples for 3 min and load on 10% polyacrylamide gel (we recommend the use of mini-gel apparatus, such as Biorad Mini Protean II or Hoefer Mighty Small ).
    12. Fix and dry the gel as usual, and expose it overnight at room temperature (take any Saran wrap off the gel before exposing).
    13. Develop film and observe the presence of the 24 kDa and of the 89 kDa fragments in apoptotic samples.
3B. Commentary

 3B.1. Troubleshooting
 

2C. Third protocol: affinity labeling of caspases 2C.1. Materials 2C.2. Methodology
  3C. Commentary
3C.1. Troubleshooting
   2D. Fourth protocol: Western blotting  2D.1. Materials
  2D.2. Methodology
   

3D. Commentary

 

3D.1. Troubleshooting

 3.2. Key references
  2. Nicholson, D. W., and Thornberry, N. A. 1997. Caspases: killer proteases. TIBS 22: 299.

3. Margolin, N., Raybuck, S. A., Wilson, K. P., Chen, W., Fox, T., Gu, Y., and Livingstone, D. J. 1997. Substrate and inhibitor specificity of interleukin-1b-converting enzyme and related caspases. J. Biol. Chem. 272: 7223.

4. Faleiro, L., Kobayashi, R., Fearnhead, H., and Lazebnik, Y. 1997. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J. 16: 2271.

 
Appendix 1 (A1): Stock solutions

 
 

Solution
Preparation
Storage
Cell Lysis Buffer 10 mM Hepes pH 7.4, 50 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 1 mM PMSF (add immediately before use,) 2 mg/ml each leupeptin and aprotinin (add immediately before use).
Use fresh.
2x Reaction Buffer 50 mM Hepes pH 7.4, 0.2% CHAPS, 20% Glycerol, 2 mM EDTA, 10 mM DTT (add immediately before use).
Use fresh.
5x Reaction Buffer  50 mM Hepes pH 7.4, 0.5% CHAPS, 25 mM DTT (add immediately before use).
Use fresh.
MDB Buffer  50 mM NaCl, 2 mM MgCl,, 5 mM EGTA 10 mM Hepes pH 7.0, 1 mM DTT (add immediately before use).
Use fresh.
KPM Buffer  50 mM KCl, 50 mM PIPES pH 7.0, 10 mM EGTA, 1.92 mM MgCl2, 1 mM DTT, 0.1 mM PMSF (add immediately before use), 10 mg/ml cytochalasin B (add immediately before use), 2 mg/ml each chymostatin, pepstatin, leupeptin, antipain (add immediately before use).
Use fresh.
PTB Buffer  20 mM Tris/HCl, 150 mM NaCl, 0.02% Tween-20.
RT
Lysis Buffer (LB)  150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1mM EGTA, 2% Triton X-100, 0.2 mM Na3VO4, 1 mg/ml leupeptin (add immediately before use), 20 mg/ml aprotinin (add immediately before use), 0.2 mM PMSF (add immediately before use). 
Use fresh.
Buffer A 50 mM HEPES pH 7.4, 5 mM b-mercaptoethanol, 20% isopropanol.  
Transfer buffer 10 mM Tris base, 100 mM glycine, 20% methanol.  
Washing solution  Tween 20 (0.05-0.1%) in PBS.  
Blocking solution  Low-fat milk 5% in PBS.  
 

 Appendix 2 (A2): Reagents
Antibodies against caspases or substrates
Several commercial sources

Autoradiography film

Avidin-Neutralite
Molecular Probes

Biotinylated HRP
Molecular Probes

Biotin-YVAD-amk
Biosyn, Ireland

DC protein assay 500-0116
Bio-Rad

Ac-DEVD-AMC
Bachem

DEVD-AFC
Enzyme Systems Inc

DEVD-CHO
Bachem

DEVD-pNA
Bachem

ECL
Amersham

HRP-conjugated anti-Ig antibody
Several commericial sources

Immobilon PSQ membrane
Millipore

35S Methionine 1,000 Ci/mMol
Amersham

Super Signal
Pierce

TNT Coupled Reticulocyte Lysate System
Promega

Ac-YVAD-AMC
Bachem

YVAD-AFC
Enzyme Systems Inc.

YVAD-CHO
Bachem

YVAD-pNA
Bachem
 

Appendix 3 (A3): Equipment
Fluorometer or 96-well plate reader

Spectrofotometer or 96-well plate reader

SDS-PAGE apparatus mod. Mini Protean II Biorad
or
SDS-PAGE apparatus mod. Mighty Small
Hoefer

Power supply

Gel dryer

Autoradiography cassette

 Plotting apparatus