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Z-VAD-FMK

Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone) is a cell-permeable, irreversible pan caspase inhibitor that binds to the catalytic site of caspase proteases and can inhibit induction of apoptosis. For inhibition of apoptosis, Z-VAD-FMK should be added at the same time that apoptosis is induced. The peptide is O-methylated in the P1 position on aspartic acid, providing enhanced stability and increased cell permeability. Fluoromethyl ketone (FMK)-derivatized peptides act as effective irreversible inhibitors with no added cytotoxic effects.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Z-VAD-FMK stock solution
Add appropriate amount of DMSO into Z-VAD-FMK to make 2-5 mM Z-VAD-FMK stock solution.
Note     Store the unused Z-VAD-FMK stock solution at -20 °C in single use aliquots.

SAMPLE EXPERIMENTAL PROTOCOL

The following protocol can be used as a guideline and should be optimized according to your needs.
  1. Treat your samples as desired to induce apoptosis.
  2. Add Z-VAD-FMK along with the treatment of your choice.
    Note     We recommend using 10-100 µM of final concentration ofZ-VAD-FMK to inhibit caspases such as 50 µM for caspase-8.
    Note     Optimal time and concentration for incubation needs to be determined experimentally.
  3. Stain samples as you desired.
  4. Monitor the fluorescence intensity with a proper instrument. 

References

View all 8 references: Citation Explorer
Identification of active caspases using affinity-based probes.
Authors: McStay, Gavin P and Green, Douglas R
Journal: Cold Spring Harbor protocols (2014): 856-60
Induction of apoptosis in human renal cell carcinoma cells by vitamin E succinate in caspase-independent manner.
Authors: Wu, Xiu-Xian and Kakehi, Yoshiyuki and Jin, Xing-Hua and Inui, Masashi and Sugimoto, Mikio
Journal: Urology (2009): 193-9
Induction of apoptosis in renal tubular cells by histone deacetylase inhibitors, a family of anticancer agents.
Authors: Dong, Guie and Wang, Lysa and Wang, Cong-Yi and Yang, Tianxin and Kumar, M Vijay and Dong, Zheng
Journal: The Journal of pharmacology and experimental therapeutics (2008): 978-84
Metformin induces apoptosis of pancreatic cancer cells.
Authors: Wang, Luo-Wei and Li, Zhao-Shen and Zou, Duo-Wu and Jin, Zhen-Dong and Gao, Jun and Xu, Guo-Ming
Journal: World journal of gastroenterology (2008): 7192-8
Apoptosis of gastric cancer cell line MKN45 by photodynamic treatment with photofrin.
Authors: Takahira, Kenichiro and Sano, Munetaka and Arai, Hajime and Hanai, Hiroyuki
Journal: Lasers in medical science (2004): 89-94
Page updated on May 19, 2025

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Catalog Number13300
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Physical properties

Molecular weight

467.49

Solvent

DMSO

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501
Inhibition of caspase activities in Jurkat cells with pan caspase inhibitor Z-VAD-FMK (Cat.# 13300). Jurkat cells were seeded on the same day at 200,000 cells/90 &micro;L/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 &micro;M of staurosporine for 5 hours. The Z-VAD-FMK (50 uM) was added and co-incubated with staurosporine. The caspase-8 activity was detected with Cell Meter&trade; Caspase 8 Activity Apoptosis Assay Kit (Cat.# 22816). The fluorescence intensity was measured at Ex/Em = 540/620 nm (Cutoff = 610 nm) with Flexstation 3 fluorescence microplate reader (Molecular Devices).
Inhibition of caspase activities in Jurkat cells with pan caspase inhibitor Z-VAD-FMK (Cat.# 13300). Jurkat cells were seeded on the same day at 200,000 cells/90 &micro;L/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 &micro;M of staurosporine for 5 hours. The Z-VAD-FMK (50 uM) was added and co-incubated with staurosporine. The caspase-8 activity was detected with Cell Meter&trade; Caspase 8 Activity Apoptosis Assay Kit (Cat.# 22816). The fluorescence intensity was measured at Ex/Em = 540/620 nm (Cutoff = 610 nm) with Flexstation 3 fluorescence microplate reader (Molecular Devices).
Inhibition of caspase activities in Jurkat cells with pan caspase inhibitor Z-VAD-FMK (Cat.# 13300). Jurkat cells were seeded on the same day at 200,000 cells/90 &micro;L/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 &micro;M of staurosporine for 5 hours. The Z-VAD-FMK (50 uM) was added and co-incubated with staurosporine. The caspase-8 activity was detected with Cell Meter&trade; Caspase 8 Activity Apoptosis Assay Kit (Cat.# 22816). The fluorescence intensity was measured at Ex/Em = 540/620 nm (Cutoff = 610 nm) with Flexstation 3 fluorescence microplate reader (Molecular Devices).
Gallery Image 2

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
Caspases
Ac-DEVD-AFC *CAS 201608-14-2*
Ac-DEVD-AMC *CAS 169332-61-0*
Ac-DEVD-aminoluciferin