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Z-IETD-D-Cys

Caspase 8 probe (z-IETD-D-Cys)

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Probe for caspase activity

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Introduction

Caspase 3/7 cleavage of the DEVD-D-Cys peptide
Figure 1: Following caspase 8 cleavage of the IETD-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.

The Caspase 8 quantification kit is a bioluminescent assay that measures caspase 8 activities in vitro, as well as in vivo using the split-luciferin technology. Caspase 8 is cysteine/aspartic acid-specific protease and play key effector roles in apoptosis in mammalian cells1–2.

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 8 specific peptidic IETD 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 8 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 8.

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 8 quantification kit is also more sensitive than fluorescence-based caspase assays. The assay uses a substrate containing the IETD sequence that is selective for caspase 8.3-4

This technology has been developed to measure caspase 8 acitivity in live cells but also in living animals. The caspase 8 quantification kit provide a very high signal to background ratio allowing sensitive detection of caspase 8 activity.

The assay provides a luminogenic caspase-8 substrate, which contains the tetrapeptide sequence linked to D-Cysteine Z-Ile-Glu-Thr-Asp-D-Cys (Z-IETD-(D-Cys)), and 6-amino-2-cyanobenzothiazole (NH2-CBT).

Material Amount Storage Stability
Caspase 8 quantification 1 mg
  • -20° C
  • Desiccate
  • Protect from light
When stored as directed, reactive probes are stable for at least 3 months
5 mg
10 mg
1 mg is sufficient for a 96-well assay

Immediately before use dissolve IETD-D-Cys peptide in PBS and NH2-CBT in sterile DMSO. Once reconstituted, reagents should be stored at -20°C protected from light. For best results, avoid multiple freeze-thaw cycle.

Experimental Protocols

In Vivo assay

Example of in vivo caspase 8 assay using luciferase-expressing mice.

Anesthetized mice were treated IP with LPS and D-GalN. 6 hours after treatment, zIETD-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 reach your needs.

  1. Earnshaw, W. et al. (1999) Mammalian caspases: Structure, activation, substrates, and functions during apoptosis. Ann. Rev. Biochem. 68, 383-424.
  2. Muzio, M. et al. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817-27.
  3. Zhang, J.H., Chung, T.D. and Oldenburg, K. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67-73.
  4. Thornberry, N.A., Chapman, K.T. and Nicholson, D.W. (2000) Determination of caspase specificities using a peptide combinatorial library. Methods Enzymol. 322, 100-10.

Caspase 3/7 & 8 probe

  • Split-luciferin technology to monitor and quantify caspase activity in vivo.
  • Use in cells and animals expressing luciferase
  • Available today at a very reasonable price
A Biocompatible in Vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice. ACS Chem. Biol., 2013, 8 (5), pp 987–999
Caspase
Reproduced from ACS Chem. Biol., 2013, 8(5), 987-999. Copyright 2013 American Chemical Society.
Color

white powder

Molecular Weight

713.76 g.mol-1

Formula

C30H43N5O13S

Shipping Condition

Dry Ice

Regulatory Statement

For Research Use Only. Not for use in diagnostic procedures.

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