Caspase-3 Colorimetric Assay Kit

Details for Product No. ABIN411828
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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
(4), (5), (5), (3), (5), (10), (13), (4), (12), (7), (7), (14), (1)
Application
Biochemical Assay (BCA)
Pubmed 14 references available
Quantity 100 tests
Options
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Catalog No. ABIN411828
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Purpose Convenient, Highly-Sensitive Assay
Sample Type Cell Samples, 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
Background Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells.
Research Area Apoptosis/Necrosis, Autophagy
Comment

Category: Apoptosis

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.

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: Moriwaki, Shinzaki, Miyoshi: "GDP-mannose-4,6-dehydratase (GMDS) Deficiency Renders Colon Cancer Cells Resistant to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor- and CD95-mediated Apoptosis by Inhibiting Complex II Formation." in: The Journal of biological chemistry, Vol. 286, Issue 50, pp. 43123-33, 2011 (PubMed).

Li, Tong, Maimaitiyiming et al.: "Over-expression of cGMP-dependent protein kinase I (PKG-I) attenuates ischemia reperfusion induced kidney injury." in: American journal of physiology. Renal physiology, 2011 (PubMed).

Hochstrasser, Hohsfield, Sperner-Unterweger et al.: "β-Amyloid induced effects on cholinergic, serotonergic, and dopaminergic neurons is differentially counteracted by anti-inflammatory drugs." in: Journal of neuroscience research, 2012 (PubMed).

Zhang, Zhai, Li et al.: "Anti-tumor selectivity of a novel Tubulin and HSP90 dual-targeting inhibitor in non-small cell lung cancer models." in: Biochemical pharmacology, 2013 (PubMed).

Merino, Singla: "Notch-1 mediated cardiac protection following embryonic and induced pluripotent stem cell transplantation in doxorubicin-induced heart failure." in: PLoS ONE, Vol. 9, Issue 7, pp. e101024, 2014 (PubMed).

Chang, Shen, Kang et al.: "Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways." in: Toxicology and applied pharmacology, 2014 (PubMed).

General Defaux, Zurich, Honegger et al.: "Inflammatory responses in aggregating rat brain cell cultures subjected to different demyelinating conditions." in: Brain research, 2010 (PubMed).

Senthilkumar, Elumalai, Arunkumar et al.: "Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)." in: Molecular and cellular biochemistry, 2010 (PubMed).

Ark, Ozdemir, Polat: "Ouabain-induced apoptosis and Rho kinase: a novel caspase-2 cleavage site and fragment of Rock-2." in: Apoptosis : an international journal on programmed cell death, 2010 (PubMed).

Mao, Nie, Tsu et al.: "White Tea Extract Induces Apoptosis in Non-Small Cell Lung Cancer Cells: the Role of Peroxisome Proliferator-Activated Receptor-{gamma} and 15-Lipoxygenases." in: Cancer prevention research (Philadelphia, Pa.), Vol. 3, Issue 9, pp. 1132-40, 2010 (PubMed).

Nishikawa, Tsuno, Okaji et al.: "The inhibition of autophagy potentiates anti-angiogenic effects of sulforaphane by inducing apoptosis." in: Angiogenesis, Vol. 13, Issue 3, pp. 227-38, 2010 (PubMed).

Zauli, Voltan, Bosco et al.: "Dasatinib plus Nutlin-3 shows synergistic anti-leukemic activity in both p53wild-type and p53mutated B chronic lymphocytic leukemias by inhibiting the Akt pathway." in: Clinical cancer research : an official journal of the American Association for Cancer Research, 2010 (PubMed).

Heo, Oh, Kho et al.: "ERK mediates anti-apoptotic effect through phosphorylation and cytoplasmic localization of p21(Waf1/Cip1/Sdi) in response to DNA damage in normal human embryonic fibroblast (HEF) cells." in: Molecular biology reports, 2010 (PubMed).

Zhao, Huai, Jin et al.: "Quinoxaline derivative of oleanolic acid inhibits osteoclastic bone resorption and prevents ovariectomy-induced bone loss." in: Menopause (New York, N.Y.), 2011 (PubMed).

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