Analysis of Caspase Activation During Apoptosis

Scott H. Kaufmann1, Timothy J. Kottke1, L. Miguel Martins2, Alexander J. Henzing3, William C. Earnshaw3

1 Mayo Clinic, Rochester, Minnesota, 2 Imperial Cancer Research Fund, London, United Kingdom, 3 University of Edinburgh, Edinburgh, Scotland, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 18.2
DOI:  10.1002/0471143030.cb1802s11
Online Posting Date:  August, 2001
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Abstract

This unit describes three methods for the detection of caspase activation as cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are provided using suitable substrates. Because the various low‐molecular‐weight substrates available for these assays are not selective, however, the assays do not accurately distinguish between various caspases. Immunoblotting is described for following the activation of specific caspases. When coupled with subcellular fractionation, this method can provide large amounts of temporal and spatial information about caspase activation. Finally, affinity labeling protocols are provided for detecting active caspases in whole‐cell lysates or subcellular fractions.

     
 
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Table of Contents

  • Basic Protocol 1: Enzymatic Assays for Caspase Activity
  • Basic Protocol 2: Detection of Caspase Activation by Immunoblotting
  • Alternate Protocol 1: Cell Lysis with Guanidine Hydrochloride for Immunoblotting
  • Support Protocol 1: Removing (Stripping) Primary and Secondary Antibodies from Blots
  • Basic Protocol 3: Labeling and Detecting Active Caspases Using Biotinylated Substrate Analogs
  • Alternate Protocol 2: In Vitro Activation of Caspases in Naive Lysates Followed by Affinity Labeling
  • Support Protocol 2: Controls for Specificity of Affinity‐Labeled Active Caspases
  • Support Protocol 3: Stripping Membrane in the Presence of d‐Biotin for Reprobing with Antibody
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enzymatic Assays for Caspase Activity

  Materials
  • Cells of interest and appropriate medium
  • Apoptosis‐inducing stimulus
  • CMF‐DPBS ( appendix 2A), ice cold
  • Lysis buffer (see recipe), 4°C
  • 0.5 M EDTA, pH 7.4 (see recipe)
  • 1 M dithiothreitol (DTT; appendix 2A)
  • 5 mM EDTA (pH 7.4)/1 mM DTT in lysis buffer, ice cold
  • 20 mM (14.6 mg/ml) acetyl‐Asp‐Glu‐Val‐Asp‐7‐amino‐4‐trifluoromethylcoumarin (acetyl‐DEVD‐AFC; Biomol), or other fluorogenic or chromogenic caspase substrate, in dimethyl sulfoxide (DMSO)
  • HEPES/CHAPS buffer (see recipe), room temperature and ice cold
  • 10 mM (4.6 mg/ml) 7‐amino‐4‐trifluoromethylcoumarin (free AFC; Sigma), or appropriate standard for other substrate, in DMSO
  • Cell scraper (optional)
  • 2‐ml (total volume) tight‐fitting Dounce homogenizers
  • Beckman TL100 ultracentrifuge and TL100.2 rotor, or equivalent, 4°C, and appropriate ultracentrifuge tubes
  • Fluorometer
  • Additional reagents and equipment for Ficoll‐Hypaque density sedimentation (unit 2.2), cell trypsinization (for adherent cell lines only; unit 1.1), trypan blue staining to detect lysed cells (unit 1.1), and determination of protein concentration ( appendix 3B)
NOTE: Fluorogenic and chromogenic caspase substrates are available from a number of suppliers, including Bachem Bioscience, Biomol Research Laboratories, Calbiochem‐Novabiochem, Molecular Probes, and Osaka Peptide Institute. Stock solutions for substrates and standards are prepared as 20 mM solutions in DMSO and stored in aliquots for up to 1 year at −20°C.

Basic Protocol 2: Detection of Caspase Activation by Immunoblotting

  Materials
  • CMF‐DPBS ( appendix 2A), ice cold
  • Serum‐free tissue culture medium (appropriate for cells of interest), ice cold, optional
  • SDS sample buffer (see recipe)
  • Fast green dye solution: 0.1% (w/v) fast green FCF/20% (v/v) methanol/5% (v/v) acetic acid
  • Fast green destain: 20% (v/v) methanol/5% (v/v) acetic acid
  • Blocking buffer (see recipe)
  • Anti‐caspase primary antibody (Table 18.2.1)
  • PBS‐T (see recipe)
  • Appropriate secondary antibody conjugated to horseradish peroxidase (HRP), alkaline phosphatase (AP), or a radiolabel
  • 3% (w/v) nonfat dry milk in CMF‐DPBS
  • Enhanced chemiluminescence reagents (e.g., ECL from Amersham Pharmacia Biotech), for HRP‐conjugated secondary antibodies
  • X‐ray film
  • Cell scraper (optional)
  • Sonicator equipped with microprobe (e.g., Branson)
  • 70° or 100°C water bath or heating block
  • Additional reagents and equipment for inducing apoptosis (see protocol 1), trypsinizing and counting cells (optional; unit 1.1), SDS‐polyacrylamide gel electrophoresis (unit 6.1), and electrophoretic transfer of polypeptides to a solid support (unit 6.2)
    Table 8.2.1   Materials   Selected Properties of Human Caspases a   Selected Properties of Human Caspases

    New name Old name(s) Molecular weight (kDa) b Preferred small substrates c Antibody suppliers d
    Pr Lg Sm
    Caspase‐1 ICE 45 24 14 YEVD/X WEHD/X BP, ORP, UB
    20 10
    Caspase‐2 Ich‐1, NEDD2 L 48 32 14 VDVAD/X DEHD/X BP, BTL, ORP, UB
    18 12
    Caspase‐3 CPP32, YAMA, Apopain 32 20 12 DMQD/X DEVD/X BP, BTL, CI, ORP, UB
    17
    Caspase‐4 Tx, Ich‐2, ICE relII LEVD/X (W/L)EHD/X BP, ORP
    Caspase‐5 ICE relIII, Ty Unknown (W/L)EHD/X CN
    Caspase‐6 Mch2 34 21 13 VEID/X VEHD/X BP, CI, CN, NEB, UB
    18 11
    Caspase‐7 Mch3, CMH‐1, ICE‐LAP3 34 20 12 DEVD/X DEVD/X BP, BTL, NEB, ORP
    Caspase‐8 Mch5, FLICE,MACH 53 43 12 IETD/X LETD/X BP, CI, CN, NEB, ORP
    55 18 11
    Caspase‐9 ICE‐LAP6, Mch6 50 37 12 Unknown LEDH/X BP, CI, CN, NEB, UB
    Caspase‐10 Mch4, FLICE‐2 55 43 12 IEAD/X Unknown CI, CN, NEB, UB
    17
    Caspase‐13 ERICE Unknown Unknown
    Caspase‐14 MICE 29 18 10 Unknown Unknown BTL, ORP

     aModified from Earnshaw et al. ( ).
     bThe appearance of multiple entries indicates partially processed and fully processed large (Lg) and small (Sm) subunits that result from sequential cleavage at the C‐terminal end of the large subunit followed by removal of the linker peptide from the small subunit and the prodomain (Pr) from the large subunit. A blank in this column indicates that the molecular weight of the processed forms has not been reported.
     cThe left and right columns indicate the preferred low‐molecular‐weight substrate specificity reported by two groups: Talanian et al. ( ) and Thornberry et al. ( ), respectively. It is important to note, however, that additional factors also affect caspase cleavage of full‐length polypeptides. Not all sites conforming to the indicated sequences are cleaved, perhaps due to limited accessibility. Conversely, polypeptides are sometimes cleaved at sites that would not be predicted based on analysis of the tetrapeptide preferences indicated in this table (Samejima et al., ).
     dAbbreviations: BP, BD PharMingen; BTL, BD Transduction Laboratories; CI, Chemicon International; CN, Calbiochem‐Novabiochem; NEB, New England Biolabs; ORP, Oncogene Research Products; UB, Upstate Biotechnology.

Alternate Protocol 1: Cell Lysis with Guanidine Hydrochloride for Immunoblotting

  • Guanidine hydrochloride lysis buffer (see recipe)
  • 100 mM PMSF ( appendix 2A)
  • 2‐Mercaptoethanol (2‐ME)
  • 1.54 M (285 mg/ml) iodoacetamide in guanidine hydrochloride lysis buffer, prepared fresh
  • 4 M urea (see recipe)/50 mM Tris⋅Cl, pH 7.4 at 4°C ( appendix 2A)
  • 0.1% (w/v) SDS
  • 1‐cm dialysis tubing (MWCO 8000 to 10,000), double knotted at one end, and dialysis clips
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)
CAUTION: 2‐ME has a strong odor and its use is confined to the hood in some laboratories.NOTE: All steps involving iodoacetamide should be performed under subdued light because of the light sensitivity of the carbon‐iodine bond.

Support Protocol 1: Removing (Stripping) Primary and Secondary Antibodies from Blots

  Materials
  • Nitrocellulose or PVDF membrane with bound antibodies (see protocol 2 or protocol 3)
  • Blot erasure buffer (see recipe)
  • CMF‐DPBS ( appendix 2A)
  • Resealable plastic bags
  • 65°C water bath

Basic Protocol 3: Labeling and Detecting Active Caspases Using Biotinylated Substrate Analogs

  Materials
  • Incomplete KPM buffer (see recipe), 4°C
  • Complete KPM buffer (see recipe), 4°C
  • 100 µM N‐(Nα‐benzyloxycarbonylglutamyl‐Nɛ‐biotinyllysyl)aspartic acid ([2,6‐dimethylbenzoyl]oxy)methylketone [zEK(bio)D‐aomk; Osaka Peptide Institute] in dimethyl sulfoxide (DMSO), stored in aliquots up to 2 years at −80°C
  • 3× SDS sample buffer (see recipe)
  • 5% (w/v) nonfat dry milk in recipePBS‐T
  • PBS‐T (see recipe)
  • Peroxidase‐coupled streptavidin (e.g., Amersham Pharmacia Biotech)
  • Enhanced chemiluminescence reagents (e.g., ECL; Amersham Pharmacia Biotech)
  • Cell scraper (optional)
  • 8 × 34–mm polycarbonate ultracentrifuge tubes (e.g., Beckman)
  • Beckman Optima TLX tabletop ultracentrifuge and TL100.1 rotor, or equivalent, 4°C
  • Additional reagents and equipment for inducing apoptosis (see protocol 1), cell trypsinization (optional; unit 1.1), protein determination ( appendix 3B), SDS‐polyacrylamide gel electrophoresis (unit 6.1), and electrophoretic transfer of polypeptides to a solid support (unit 6.2)

Alternate Protocol 2: In Vitro Activation of Caspases in Naive Lysates Followed by Affinity Labeling

  • Incomplete KHM buffer (see recipe), 4°C
  • Complete KHM buffer (see recipe), 4°C
  • 5 µg/ml active caspase‐8 (BD PharMingen) in complete KHM buffer or 5 mg/ml cytochrome c (Sigma) in complete KHM buffer
  • 10 mM dATP (Sigma) in complete KHM, pH 7.4, optional
  • 100 µM N‐(Nα‐benzyloxycarbonylglutamyl‐Nɛ‐biotinyllysyl)aspartic acid ([2,6‐dimethylbenzoyl]oxy)methylketone [zEK(bio)D‐aomk; Osaka Peptide Institute] in dimethyl sulfoxide (DMSO), stored in aliquots up to 2 years at −80°C

Support Protocol 2: Controls for Specificity of Affinity‐Labeled Active Caspases

  • Membrane containing affinity‐labeled caspases (see protocol 5)
  • 2 mM d‐biotin (Sigma) in PBS‐T
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Figures

Videos

Literature Cited

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Key References
   Enari, M., Talanian, R.V., Wong, W.W., and Nagata, S. 1996. Sequential activation of ICE‐like and CPP32‐like proteases during fas‐mediated apoptosis. Nature 380:723‐726.
  This paper illustrates the use of different tetrapeptide substrates to demonstrate the sequential activation of multiple caspases during apoptosis.
   Schlegel, J., Peters, I., Orrenius, S., Miller, D.K., Thornberry, N.A., Yamin, T.T., and Nicholson, D.W. 1996. CPP32/apopain is a key interleukin 1 beta converting enzyme‐like protease involved in fas‐mediated apoptosis. J. Biol. Chem. 271:1841‐1844.
  This paper illustrates one of the first uses of immunoblotting to demonstrate caspase activation during apoptosis.
   Takahashi et al., 1996b. See above.
  This paper describes the first use of affinity labeling to detect active caspases in extracts from apoptotic cells.
Internet Resources
  www.peptide.co.jp
  The Web site of the Osaka Peptide Institute contains a variety of caspase substrates as well as acycloxymethyl ketones that can be used as affinity labels.
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