Caspase-3 Colorimetric Assay Kit

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Target Name (Antigen)
Synonyms
CC3, CG14902, Cas3, Casp 3, Casp3, Caspase-3, DECAY, Decay, Dmel\\CG14902, Drosophila executioner caspase related to Apopain/Yama, caspase 3, caspase-3, CG7788, DRICE, Dmel\\CG7788, DrICE, DrIce, Dric ... show more
CC3, CG14902, Cas3, Casp 3, Casp3, Caspase-3, DECAY, Decay, Dmel\\CG14902, Drosophila executioner caspase related to Apopain/Yama, caspase 3, caspase-3, CG7788, DRICE, Dmel\\CG7788, DrICE, DrIce, Drice, ICE, crice, drICE, drIce, drice, ice, A830040C14Rik, AC-3, Apopain, CPP32, Lice, Yama, mldy, CPP32B, SCA-1, xcpp32 show less
Reactivity
Mammalian
(3), (6), (4), (2), (4), (12), (13), (3), (13), (6), (7), (14), (1)
Application
Biochemical Assay (BCA)
Pubmed 21 references available
Quantity 100 tests
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Purpose Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. The Caspase-3/CPP32 Colorimetric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400- or 405 nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in CPP32 activity.
Sample Type Cell Lysate, Tissue Lysate
Detection Method Colorimetric
Specificity The Caspase-3/CPP32 Colorimetric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400- or 405-nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in CPP32 activity.
Characteristics Caspase-3,CPP32 Activity Colorimetric Assay Kit: Simple & Convenient Colorimetric Assay to Measure Caspase-3,CPP32 Activity within 1-2 hrs.
Components Cell Lysis buffer
2X Reaction Buffer
DEVD-pNA (4 mM)
DTT (1 M)
Dilution Buffer
Alternative Name Caspase-3 (CASP3 ELISA Kit Abstract)
Background Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells.
Research Area Apoptosis/Necrosis, Autophagy
Application Notes Detect early/middle stages of apoptosis, differentiate apoptosis from necrosis.
Comment

Absorbance (400 or 405 nm)
Simple one-step procedure, takes 1-2 hours
Fast and convenient
Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in CPP32 activity.

Assay Time 1 - 2 h
Protocol A. General Considerations Aliquot enough 2X Reaction Buffer for the number of assays to be performed. Add DTT to the 2X Reaction Buffer immediately before use (10 mM final concentration: add 10 µL of 1.0 M DTT stock per 1 mL of 2X Reaction Buffer). Protect DEVD-pNA from light. B.
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5 x 106 cells.
3. Resuspend cells in 50 µL of chilled Cell Lysis Buffer and incubate cells on ice for 10 minutes.
4. Centrifuge for 1 min in a microcentrifuge (10,000 x g).
5. Transfer supernatant (cytosolic extract) to a fresh tube and put on ice for immediate assay or aliquot and store at -80 °C for future use.
6. Assay protein concentration.
7. Dilute 50-200 µg protein to 50 µL Cell Lysis Buffer for each assay.
8. Add 50 µL of 2X Reaction Buffer (containing 10 mM DTT) to each sample.
9. Add 5 µL of the 4 mM DEVD-pNA substrate (200 µM final conc.) and incubate at 37 °C for 1-2 hour.
10. Read samples at 400- or 405-nm in a microtiter plate reader, or spectrophotometer using a 100- µL micro quartz cuvet (Sigma), or dilute sample to 1 mL with Dilution Buffer and using regular cuvet (note: Dilution of the samples proportionally decreases the reading). You may also perform the entire assay directly in a 96-well plate. Fold-increase in CPP32 activity can be determined by comparing these results with the level of the uninduced control. Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in CPP32 activity.
Restrictions For Research Use only
Storage -20 °C
Storage Comment Store kit at -20 °C (Store Lysis Buffer, Reaction Buffer, and Dilution Buffer at 4 °C after opening).
Expiry Date 12 months
Product cited in: Salminen, Dahle, Moen et al.: "Intracoronary insulin administered at reperfusion in a porcine model of acute coronary syndrome." in: European heart journal. Acute cardiovascular care, Vol. 4, Issue 3, pp. 230-40, 2015 (PubMed).

Sudan, Rupasinghe: "Quercetin-3-O-glucoside induces human DNA topoisomerase II inhibition, cell cycle arrest and apoptosis in hepatocellular carcinoma cells." in: Anticancer research, Vol. 34, Issue 4, pp. 1691-9, 2014 (PubMed).

Fang, Moore, Bai et al.: "Hydrogen peroxide enhances radiation-induced apoptosis and inhibition of melanoma cell proliferation." in: Anticancer research, Vol. 33, Issue 5, pp. 1799-807, 2013 (PubMed).

Rana, Bera, Bhattacharya et al.: "Characterization of arsenic-induced cytotoxicity in liver with stress in erythrocytes and its reversibility with Pleurotus florida lectin." in: Toxicology and industrial health, Vol. 31, Issue 2, pp. 108-22, 2015 (PubMed).

Terasaki, Won, Makino: "The C-terminal region of Rift Valley fever virus NSm protein targets the protein to the mitochondrial outer membrane and exerts antiapoptotic function." in: Journal of virology, Vol. 87, Issue 1, pp. 676-82, 2012 (PubMed).

Siddiqui, Kumar, Kashyap et al.: "Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells." in: Human & experimental toxicology, Vol. 31, Issue 4, pp. 336-45, 2012 (PubMed).

Li, Wang, Xue et al.: "Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5." in: Journal of virology, Vol. 86, Issue 3, pp. 1328-38, 2012 (PubMed).

Fang, Herrick, Nicholl: "A possible role for perforin and granzyme B in resveratrol-enhanced radiosensitivity of prostate cancer." in: Journal of andrology, Vol. 33, Issue 4, pp. 752-60, 2012 (PubMed).

Bakheet, Attia, Al-Rasheed et al.: "Salubrious effects of dexrazoxane against teniposide-induced DNA damage and programmed cell death in murine marrow cells." in: Mutagenesis, Vol. 26, Issue 4, pp. 533-43, 2011 (PubMed).

Toyonaga, Tsuruya, Ikeda et al.: "Spironolactone inhibits hyperglycemia-induced podocyte injury by attenuating ROS production." in: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, Vol. 26, Issue 8, pp. 2475-84, 2011 (PubMed).

Kannan, Colorado, Reino et al.: "Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 300, Issue 5, pp. G853-61, 2011 (PubMed).

Zhong, Wang, Guo et al.: "Protein S protects neurons from excitotoxic injury by activating the TAM receptor Tyro3-phosphatidylinositol 3-kinase-Akt pathway through its sex hormone-binding globulin-like region." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 30, Issue 46, pp. 15521-34, 2010 (PubMed).

Witko-Sarsat, Mocek, Bouayad et al.: "Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival." in: The Journal of experimental medicine, Vol. 207, Issue 12, pp. 2631-45, 2010 (PubMed).

Fang, Yu, Ellis et al.: "Comparison of sensitivity of Th1, Th2, and Th17 cells to Fas-mediated apoptosis." in: Journal of leukocyte biology, Vol. 87, Issue 6, pp. 1019-28, 2010 (PubMed).

Zhang, Soto, Park et al.: "Nuclear receptor SHP, a death receptor that targets mitochondria, induces apoptosis and inhibits tumor growth." in: Molecular and cellular biology, Vol. 30, Issue 6, pp. 1341-56, 2010 (PubMed).

Song, Zhang, Wang: "MicroRNA-206 targets notch3, activates apoptosis, and inhibits tumor cell migration and focus formation." in: The Journal of biological chemistry, Vol. 284, Issue 46, pp. 31921-7, 2009 (PubMed).

Su, Chi, Huang et al.: "15-deoxy-Delta12,14-prostaglandin J2 up-regulates death receptor 5 gene expression in HCT116 cells: involvement of reactive oxygen species and C/EBP homologous transcription factor gene transcription." in: Molecular cancer therapeutics, Vol. 7, Issue 10, pp. 3429-40, 2008 (PubMed).

Yokomaku, Sugimoto, Kume et al.: "Asialoerythropoietin prevents contrast-induced nephropathy." in: Journal of the American Society of Nephrology : JASN, Vol. 19, Issue 2, pp. 321-8, 2008 (PubMed).

Singla, McDonald: "Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells." in: American journal of physiology. Heart and circulatory physiology, Vol. 293, Issue 3, pp. H1590-5, 2007 (PubMed).

Yasuda, Fukuo, Sun et al.: "Apop-1, a novel protein inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis." in: The Journal of biological chemistry, Vol. 281, Issue 33, pp. 23899-907, 2006 (PubMed).

Zhou, Hu, Ma et al.: "Nucleocytoplasmic trafficking of the Syk protein tyrosine kinase." in: Molecular and cellular biology, Vol. 26, Issue 9, pp. 3478-91, 2006 (PubMed).

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Catalog No. ABIN411828
363.00 $
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100 tests
363.00 $

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