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Caspase-3 Colorimetric Assay Kit Kit

BCA Reactivity: Mammalian Colorimetric Cell Lysate, Tissue Samples
Pubmed (99)
Catalog No. ABIN411829
$535.00
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  • Target
    Caspase 3 (CASP3)
    Reactivity
    • 11
    • 9
    • 8
    • 6
    • 3
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    Mammalian
    Detection Method
    Colorimetric
    Application
    Biochemical Assay (BCA)
    Purpose
    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 Samples
    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
  • Application Notes
    Detect early/middle stages of apoptosis, differentiate apoptosis from necrosis.
    Comment

    Further details regarding sample type: Cell and tissue lysates

    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
  • Chandravanshi, Bhonde: "Small molecules exert anti-apoptotic effect and reduce oxidative stress augmenting insulin secretion in stem cells engineered islets against hypoxia." in: European journal of pharmacology, Vol. 791, pp. 424-432, 2016 (PubMed).

    Saing, Wei, Tseng: "Ergothioneine represses inflammation and dysfunction in human endothelial cells exposed to oxidized low-density lipoprotein." in: Clinical and experimental pharmacology & physiology, 2015 (PubMed).

    Fernando, Rodriguez-Malave, Waters, Yan, Casero, Basso, Pigazzi, Rao: "LncRNA Expression Discriminates Karyotype and Predicts Survival in B-Lymphoblastic Leukemia." in: Molecular cancer research : MCR, Vol. 13, Issue 5, pp. 839-51, 2015 (PubMed).

    Griffiths, Doe, Jijiwa, Van Ry, Cruz, de la Vega, Ramos, Burkin, Matter: "Bit-1 is an essential regulator of myogenic differentiation." in: Journal of cell science, Vol. 128, Issue 9, pp. 1707-17, 2015 (PubMed).

    Lee, Morales, Slaga, Kim: "Activation of T-cell protein-tyrosine phosphatase suppresses keratinocyte survival and proliferation following UVB irradiation." in: The Journal of biological chemistry, Vol. 290, Issue 1, pp. 13-24, 2015 (PubMed).

    Rana, Bera, Bhattacharya, Das, Pan, Das: "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).

    Salminen, Dahle, Moen, Jonassen, Haaverstad, Matre, Grong: "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).

    Giorgi, Bonora, Sorrentino, Missiroli, Poletti, Suski, Galindo Ramirez, Rizzuto, Di Virgilio, Zito, Pandolfi, Wieckowski, Mammano, Del Sal, Pinton: "p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 6, pp. 1779-84, 2015 (PubMed).

    Yoo, Pedigo, Guzman, Correa-Medina, Wei, Villarreal, Mitrofanova, Leclercq, Faul, Li, Kretzler, Nelson, Lehto, Forsblom, Groop, Reiser, Burke, Fornoni, Merscher: "Sphingomyelinase-like phosphodiesterase 3b expression levels determine podocyte injury phenotypes in glomerular disease." in: Journal of the American Society of Nephrology : JASN, Vol. 26, Issue 1, pp. 133-47, 2015 (PubMed).

    Chang, Shen, Kang, Sher, Sheu, Chang, Lee: "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).

    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).

    Baker, Maitra, Geng, Li: "Molecular and cellular mechanisms responsible for cellular stress and low-grade inflammation induced by a super-low dose of endotoxin." in: The Journal of biological chemistry, Vol. 289, Issue 23, pp. 16262-9, 2014 (PubMed).

    Jin, Lee, Min, Smith, Hwang, Whang, Kim, Kim, Lee: "Transcriptional and posttranslational regulation of insulin-like growth factor binding protein-3 by Akt3." in: Carcinogenesis, Vol. 35, Issue 10, pp. 2232-43, 2014 (PubMed).

    Joshi, Mahfooz, Maurya, Kumar, Basanna, Kaur, Hanif, Jha: "PARP1 during embryo implantation and its upregulation by oestradiol in mice." in: Reproduction (Cambridge, England), Vol. 147, Issue 6, pp. 765-80, 2014 (PubMed).

    Kang, Ko, Moon, Yoo, Jung, Kim, Koh, Kim, Kim: "Osteoprotegerin expressed by osteoclasts: an autoregulator of osteoclastogenesis." in: Journal of dental research, Vol. 93, Issue 11, pp. 1116-23, 2014 (PubMed).

    Nie, Zhang, Zhao, Liu, Niu: "Involvement of mitochondrial pathway in benzo[a]pyrene-induced neuron apoptosis." in: Human & experimental toxicology, Vol. 33, Issue 3, pp. 240-50, 2014 (PubMed).

    Park, Chang, Lee, Lee, Kang, Chun, Woo, Kong, Ko, Suzuki, Song, Park: "Targeting of miR34a-NOTCH1 axis reduced breast cancer stemness and chemoresistance." in: Cancer research, Vol. 74, Issue 24, pp. 7573-82, 2014 (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).

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

    Bersell, Choudhury, Mollova, Polizzotti, Ganapathy, Walsh, Wadugu, Arab, Kühn: "Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death." in: Disease models & mechanisms, Vol. 6, Issue 6, pp. 1459-69, 2013 (PubMed).

  • Target
    Caspase 3 (CASP3)
    Alternative Name
    Caspase-3 (CASP3 ELISA Kit Abstract)
    Synonyms
    CPP32, CPP32B, SCA-1, A830040C14Rik, AC-3, Apopain, CC3, Caspase-3, Lice, Yama, mldy, xcpp32, casp3, zgc:100890, CASP-3, caspase-3, caspase 3, caspase 3 S homeolog, caspase 3, apoptosis-related cysteine peptidase a, caspase 3, apoptosis-related cysteine peptidase, CASP3, Casp3, casp3.S, casp3a
    Background
    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.
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