Z-DEVD-AFC
Z-DEVD-AFC is a fluorogenic substrate for caspase 3, a protease that is rapidly activated when cells are exposed to apoptotic conditions and that cleaves poly(ADP-ribose) polymerase. This substrate is hydrolyzed by caspase 3 to generate highly fluorescent 7-amido-4-trifluoromethylcoumarin (AFC).
Example protocol
AT A GLANCE
Important notes
It is important to store at <-15 °C and should be stored in cool, dark place.
It can be used within 12 months from the date of receipt.
SAMPLE EXPERIMENTAL PROTOCOL
Following protocol only provides a guideline, and should be modified according to your specific needs.
General Solution Caspase Assays Using AMC, AFC, pNA, R110 and ProRed Substrates
- Prepare a 10 mM stock solution in DMSO.
- Prepare a 2X caspase substrate (50 µM) assay solution as the following: 50 µL substrate stock solution, 100 µL DTT (1M), 400 µL EDTA (100 mM), 10 mL Tris Buffer (20 mM), pH =7.4.
- Mix equal volume of the caspase standards or samples with 2X caspase substrate assay solution, and incubate the solutions at room temperature for at least 1 hour.
- Monitor the fluorescence using a fluorescence microplate reader, or absorbance using an absorbance microplate reader.
Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes
- Prepare a 2-5 mM stock solution in DMSO.
- Treat cells as desired.
- Prepare a 2X permeable caspase substrate (20 µM) assay solution by diluting the DMSO stock solution (from Step 2.1) in Hanks with 20 mM Hepes buffer (HHBS).
- Mix equal volume of the treated cells with 2X caspase substrate assay solution (from Step 2.3), and incubate the cells in a 37°C, 5% CO2 incubator for at least1 hour.
- Wash the cells with HHBS for at least once.
- Monitor the fluorescence intensity by a flow cytometer, a fluorescence microscope or a fluorescence microplate reader.
Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes (For #13470-13476 only)
- Prepare a 250X stock solution by adding 50 µL DMSO into the vial.
- Treat cells as desired.
- Add 250 X DMSO stock solution into the cell solution at a 1:250 ratio (such as 2 µL to 500 µL cells), and incubate the cells in a 37°C, 5% CO2 incubator for 1 hour.
- Wash the cells with HHBS for at least once.
- Monitor the fluorescence intensity by flow cytometer, fluorescence microscopy or fluorescent microplate reader.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Z-DEVD-AFC to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 121.697 µL | 608.487 µL | 1.217 mL | 6.085 mL | 12.17 mL |
5 mM | 24.339 µL | 121.697 µL | 243.395 µL | 1.217 mL | 2.434 mL |
10 mM | 12.17 µL | 60.849 µL | 121.697 µL | 608.487 µL | 1.217 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
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Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
Ac-DEVD-AFC *CAS 201608-14-2* | 376 | 482 | 170001 |
Z-DEVD-AMC | 341 | 441 | - |
Z-IETD-AFC *CAS 219138-02-0* | 376 | 482 | 170001 |
Z-DEVD-aminoluciferin | 362 | 499 | 18000 |
Citations
View all 2 citations: Citation Explorer
Palmitoylation is required for TNF-R1 signaling
Authors: Zingler, Philipp and Särchen, Vinzenz and Glatter, Timo and Caning, Lotta and Saggau, Carina and Kathayat, Rahul S and Dickinson, Bryan C and Adam, Dieter and Schneider-Brachert, Wulf and Schütze, Stefan and others, undefined
Journal: Cell Communication and Signaling (2019): 1--16
Authors: Zingler, Philipp and Särchen, Vinzenz and Glatter, Timo and Caning, Lotta and Saggau, Carina and Kathayat, Rahul S and Dickinson, Bryan C and Adam, Dieter and Schneider-Brachert, Wulf and Schütze, Stefan and others, undefined
Journal: Cell Communication and Signaling (2019): 1--16
pH-Assisted surface functionalization of selenium nanoparticles with curcumin to achieve enhanced cancer chemopreventive activity
Authors: Yu, Shaoxuan and Wang, Yanru and Zhang, Wentao and Zhang, Yuhuan and Zhu, Wenxin and Liu, Yingnan and Zhang, Daohong and Wang, Jianlong
Journal: RSC Advances (2016): 72213--72223
Authors: Yu, Shaoxuan and Wang, Yanru and Zhang, Wentao and Zhang, Yuhuan and Zhu, Wenxin and Liu, Yingnan and Zhang, Daohong and Wang, Jianlong
Journal: RSC Advances (2016): 72213--72223
References
View all 67 references: Citation Explorer
In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline. Involvement of caspase cascades and IkappaBalpha phosphorylation
Authors: Lerma-Diaz JM, Hern and ez-Flores G, Dominguez-Rodriguez JR, Ortiz-Lazareno PC, Gomez-Contreras P, Cervantes-Munguia R, Scott-Algara D, Aguilar-Lemarroy A, Jave-Suarez LF, Bravo-Cuellar A.
Journal: Immunol Lett (2006): 149
Authors: Lerma-Diaz JM, Hern and ez-Flores G, Dominguez-Rodriguez JR, Ortiz-Lazareno PC, Gomez-Contreras P, Cervantes-Munguia R, Scott-Algara D, Aguilar-Lemarroy A, Jave-Suarez LF, Bravo-Cuellar A.
Journal: Immunol Lett (2006): 149
Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe
Authors: Wu X, Simone J, Hewgill D, Siegel R, Lipsky PE, He L.
Journal: Cytometry A (2006): 477
Authors: Wu X, Simone J, Hewgill D, Siegel R, Lipsky PE, He L.
Journal: Cytometry A (2006): 477
Quantitative measurement of caspase-3 activity in a living starfish egg
Authors: Sakaue M, Motoyama Y, Yamamoto K, Shiba T, Teshima T, Chiba K.
Journal: Biochem Biophys Res Commun (2006): 878
Authors: Sakaue M, Motoyama Y, Yamamoto K, Shiba T, Teshima T, Chiba K.
Journal: Biochem Biophys Res Commun (2006): 878
Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3
Authors: Miller TJ, Schneider RJ, Miller JA, Martin BP, Al-Ubaidi MR, Agarwal N, Dethloff LA, Philbert MA.
Journal: Neurotoxicology (2006): 44
Authors: Miller TJ, Schneider RJ, Miller JA, Martin BP, Al-Ubaidi MR, Agarwal N, Dethloff LA, Philbert MA.
Journal: Neurotoxicology (2006): 44
Diallyl Trisulfide Induces Apoptosis of Human Gastric Cancer Cell Line MGC803 Through Caspase-3 Pathway.
Authors: Xiao XL, Peng J, Su Q, Xiang SL, Tang GH, Huang YS, Zhou XT.
Journal: Ai Zheng (2006): 1247
Authors: Xiao XL, Peng J, Su Q, Xiang SL, Tang GH, Huang YS, Zhou XT.
Journal: Ai Zheng (2006): 1247
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