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Caspase 3-7 quantification kit

1) Z-DEVD-Cys

2) 6-Amino-2-cyanobenzothiazole (amino-CBT)

Content and storage

Material Amount Storage Stability
Caspase 3/7 kit 10 mg
  • -20 °C
  • Desiccate
  • Protect from light
When stored as directed, reactive probes are stable for at least 3 months
50 mg
100 mg

General Specifications

6-Amino-2-cyanobenzothiazole (amino-CBT)

Color:  Light yellow powder 

Molecular Weight: 175.21 g.mol-1

Formula: C8H5N3S

Cas Number  7724-12-1 

Shipping Condition: Blue Ice 

Regulatory Statement: For Research Use Only. Not for use in diagnostic procedures.

Z-DEVD-D-Cys

Color:  white powder 

Molecular Weight: 713.71 g.mol-1

Formula: C29H39N5O14S 

Shipping Condition: Blue Ice 

Regulatory Statement: For Research Use Only. Not for use in diagnostic procedures. 

Introduction

The Caspase 3/7 quantification kit is a bioluminescent assay that measures caspase 3/7 activities in vitro, as well as in vivo using the split-luciferin technology. Caspase 3/7 are cysteine/aspartic acid-specific protease and play key effector roles in apoptosis in mammalian cells1–4.

The split luciferin approach enables the generation of D-luciferin through the reaction of D-Cysteine and 6-amino-2-cyanobenzothiazole (NH2-CBT). The D-Cysteine amino acid has been caged with a caspase 3/7 specific peptidic DEVD sequence. The modification of the amino group on D-Cysteine prohibits reaction with the counterpart NH2-CBT since the 1,2-aminothiol is required for the reaction with the cyano group. After cleavage of the peptide by Caspase 3/7 free D-Cysteine is liberated and can further react with NH2-CBT to form Luciferin and then bioluminescence through oxydation by Luciferase (Figure 1). Luminescence is thus proportional to the amount of active caspase 3/7.5

One of the advantages of the split luciferin approach is improved cell penetration property of CBT derivatives in comparison to full luciferin scaffold. The Caspase 3/7 quantification kit is also more sensitive than fluorescence-based caspase assays. The assay uses a substrate containing the DEVD sequence that is selective for caspase 3 and 7.6

This technology has been developed to measure caspase 3/7 acitivity in live cells but also in living animals. The caspase 3/7 quantification kit provide a very high signal to background ratio allowing sensitive detection of caspase 3/7 activity.

The assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence linked to D-Cysteine Z-Asp-Glu-Val-Asp-D-Cys (Z-DEVD-(D-Cys)), and 6-amino-2-cyanobenzothiazole (NH2-CBT).

Figure 1: Following caspase 3/7 cleavage of the DEVD-D-Cys peptide, D-Cys is released and can further react with 6-amino-2-cyanobenzothiazole to form D-aminoluciferin, resulting in the luciferase reaction and the production of light. 

Guidelines for use

Immediately before use dissolve DEVD-D-Cys peptide in 663 μL PBS (final concentration 4.5 mg.mL-1) and NH2-CBT in 147 μL sterile DMSO (final concentration 6.8 mg.mL-1). Always prepare fresh solutions and discard at the end of the day.

Experimental Protocols

         in vivo assay      

         Example of in vivo caspase 3/7 assay using luciferase-expressing mice.

         Anesthetized mice were treated IP with LPS (100 μg.kg-1) in 50 μL PBS and D-GalN (267 mg.kg-1) in 50 μL PBS. 6 hours after treatment, DEVD-D-Cys peptide (22.6 mg/kg in 100 uL PBS) is administrated IP. After 10 minutes, NH2-CBT (6.8 mg/kg in 20 uL DMSO) is administrated IP. Mice are then imaged using IVIS spectrum and bioluminescence signal is recorded every minute for 1h.5

We recommend, optimizing concentration of reagents and incubation time in order to meet your needs. 

References

1. Lakhani, S. A., Masud, A., Kuida, K., Porter, G. A., Booth, C. J., Mehal, W. Z., Inayat, I., and Flavell, R. A. (2006) Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311, 847-851. 

2. Estaquier, J., Vallette, F., Vayssiere, J., and Mignotte, B. (2012) The mitochondrial pathways of apoptosis. Adv. Exp. Med. Biol. 942, 157-183. 

3. Miyashita, T., Okamura-Oho, Y., Mito, Y., Nagafuchi, S., and Yamada, M. (1997) Dentatorubral pallidoluysian atrophy (DRPLA) protein is cleaved by caspase-3 during apoptosis. J. Biol. Chem. 272, 29238-29242. 

4. Shin, S., Sung, B. J., Cho, Y. S., Kim, H. J., Ha, N. C., Hwang, J. I., Chung, C. W., Jung, Y. K., and Oh, B. H. (2001) An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochemistry 40, 1117-1123. 

5. Godinat A., Park H.M., Miller S.C., Cheng K., Hanahan D., Sanman L.E., Bogyo M., Yu A., Nikitin G.F., Stahl A., Dubikovskaya E.A. (2013) A Biocompatible in vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice, ACS Chem Biol. 8, 987-999. 

6. Bayascas, J.R. et al. (2002) Isolation of AmphiCASP-3/7, an ancestral caspase from amphioxus (Branchiostoma floridae). Evolutionary considerations for vertebrate caspases. Cell. Death Differ. 9, 1078–89.

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