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(Ac-LEHD)2-R110
Caspase 9 is a member of the CED-3 subfamily of the caspase family of cysteine proteases that play an essential role in the execution phase of apoptosis. These enzymes share a dominant primary specificity for cleaving bonds following aspartic acid residues. "Initiator" caspases (such as caspase 8) activate "effector" caspases, such as caspases 3 and 7. The effector caspases then cleave cellular substrates ultimately leading to the morphological changes of apoptosis. (Ac-LHED)2-R110 is a selective fluorogenic substrate for caspase 9. The caspase 9-induced hydrolysis of (Ac-LHED)2-R110 results in the release of R110 fluorophore that is detected using an excitation wavelength of ~490 nm and an emission wavelengh of ~520 nm. The assay can be run in the assay buffer consisting of 50 mM MES, pH 6.5, 10% PEG 8000, 0.1% CHAPS, 5 mM DTT, and 1 mM EDTA.
Example protocol
AT A GLANCE
Important notes
Following protocol only provides a guideline, and should be modified according to your specific needs.
PREPARATION OF STOCK SOLUTION
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.
1. (Ac-LEHD)2-R110 stock solution (10X):
Prepare a 10 mM stock solution in DMSO.
PREPARATION OF WORKING SOLUTION
Add 50 μL of substrate stock solution into 100 μL of DTT (1M), 400 μL of EDTA (100 mM), and 10 mL of Tris Buffer (20 mM), pH = 7.4, to prepare a 50 μM caspase substrate assay solution (2X).
SAMPLE EXPERIMENTAL PROTOCOL
- 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 at Ex/Em = 498/520 nm, or absorbance using an absorbance microplate reader.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
| (Ac-IETD)2-R110 | 500 | 522 | 80000 |
Citations
View all 2 citations: Citation Explorer
Mahuang Xixin Fuzi decoction ameliorates apoptosis via the mitochondrial-mediated signaling pathway in MCM cells
Authors: Yang, Jia and Sun, Qihui and Qingyun, MA and Yu, Qinhui and Liu, Xiaoyun and Liu, Yanliang and Han, Yuxiu and Yang, Yong and Rong, Rong
Journal: Journal of Ethnopharmacology (2022): 115538
Authors: Yang, Jia and Sun, Qihui and Qingyun, MA and Yu, Qinhui and Liu, Xiaoyun and Liu, Yanliang and Han, Yuxiu and Yang, Yong and Rong, Rong
Journal: Journal of Ethnopharmacology (2022): 115538
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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