Human Active Caspase-3 Quantikine ELISA Kit

(12 citations)   
  • Assay Type
    Solid Phase Sandwich ELISA
  • Format
    96-well strip plate
  • Assay Length
    3.5 hours
  • Sample Type & Volume Required Per Well
    Cell Extracts (100 uL)
  • Sensitivity
    0.1 ng/mL
  • Assay Range
    0.3 - 20 ng/mL (Cell Extracts)
  • Specificity
    Biotin-ZVKD-fmk modified natural and biotin-D-fmk modified recombinant active Caspase-3. This assay also detects mouse active Caspase-3.
  • Cross-reactivity
    < 0.5% cross-reactivity observed with available related molecules.Cross-species reactivity observed with 1 or more species tested.
  • Interference
    No significant interference observed with available related molecules.
Product Summary
The Quantikine Human active Caspase-3 Immunoassay is a 3.5 hour solid phase ELISA designed to measure human active caspase-3 in cell extracts. It contains biotinylated-inhibitor derivatized recombinant caspase-3 and a monoclonal antibody raised against caspase-3. Results obtained using natural active caspase-3 covalently bound to biotin-ZVKD-fmk inhibitor in apoptotic cell extracts showed linear curves that were parallel to the Quantikine kit standard. These results indicate that this kit can be used to determine relative mass values for natural active caspase-3.

Precision
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
Cell Extracts
Intra-Assay Precision Inter-Assay Precision
Sample123123
n202020202020
Mean1.063.097.71.063.278.25
Standard Deviation0.050.160.20.070.190.5
CV%4.75.22.66.65.86.1

Recovery

The recovery of active caspase-3 spiked to three levels throughout the range of the assay in various matrices was evaluated.

Sample Type Average % Recovery Range %
Calibrator Diluent RD5-20 (1X) (n=4) 99 98-100
Extraction Buffer (1X) (n=4) 98 97-101
Extraction Buffer (1X) (n=4) 110 104-114
Extracts from Jurkat Cells Treated w/Staurosporine (n=4) 92 87-95
Linearity
To assess the linearity of the assay, five or more samples containing and/or spiked with various concentrations of active caspase-3 in each matrix were diluted with Calibrator Diluent and then assayed. Results from typical sample dilutions are
Human Active Caspase-3 Quantikine ELISA Kit
Preparation and Storage
  • Storage
    Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: Caspase-3

Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis. They are expressed as latent zymogens and are activated by an autoproteolytic mechanism or by processing by other proteases (frequently other caspases). Human caspases can be subdivided into three functional groups: cytokine activation (caspase-1, -4, -5, and -13), apoptosis initiation (caspase-2, -8, -9, -and -10), and apoptosis execution (caspase-3, -6, and -7).

Caspases are regulated by a variety of stimili, including APAF1, CFLAR/FLIP, NOL3/ARC, and members of the inhibitor of apoptosis (IAP) family such as BIRC1/NAIP, BIRC2/cIAP-1, BIRC3/cIAP-2, BIRC4/XIAP, BIRC5/Survivin, and BIRC7/Livin. IAP activity is modulated by DIABLO/SMAC or PRSS25/HTRA2/Omi. Cell-permeable and irreversible peptide inhibitors are also available for different caspases.

    • Entrez Gene IDs
      836 (Human); 12367 (Mouse);
    • Alternate Names
      Apopain; apoptosis-related cysteine protease; CASP3; CASP-3; caspase 3, apoptosis-related cysteine peptidase; Caspase3; Caspase-3; CPP32; CPP-32; CPP32B; CPP32SREBP cleavage activity 1; Cysteine protease CPP32; EC 3.4.22; EC 3.4.22.56; LICE-1; PARP cleavage protease; procaspase3; Protein Yama; SCA-1; YAMA;
    Related Research Areas
    Assay Procedure
    Refer to the product for complete assay procedure.

    Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
    1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
    2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

    3. 100 µL Standard, Control, or Sample
    4.   Add 100 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
    5.   Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

    6. 100 µL Conjugate
    7.   Add 100 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 1 hour.
    8.   Aspirate and wash 5 times.

    9. 100 µL Substrate Solution
    10.   Add 100 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.

    11. 100 µL Stop Solution
    12.   Add 100 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.
    Citations:

    R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

    12 Citations: Showing 1 - 10
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    Species
    Sample Type
    1. Didox and resveratrol sensitize colorectal cancer cells to doxorubicin via activating apoptosis and ameliorating P-glycoprotein activity
      Sci Rep, 2016;6(0):36855.
      Species: Human
      Sample Type: Cell Culture Supernates
    2. Indole-3- carbinol enhances sorafenib cytotoxicity in hepatocellular carcinoma cells: A mechanistic study
      Sci Rep, 2016;6(0):32733.
      Species: Human
      Sample Type: Cell Culture Supernates
    3. Decidualization and syndecan-1 knock down sensitize endometrial stromal cells to apoptosis induced by embryonic stimuli.
      Authors: Boeddeker S, Baston-Buest D, Fehm T, Kruessel J, Hess A
      PLoS ONE, 2015;10(4):e0121103.
      Species: Human
      Sample Type: Cell Culture Supernates
    4. A new biocompatible and antibacterial phosphate free glass-ceramic for medical applications.
      Authors: Cabal B, Alou L, Cafini F, Couceiro R, Sevillano D, Esteban-Tejeda L, Guitian F, Torrecillas R, Moya J
      Sci Rep, 2014;4(0):5440.
      Species: Human
      Sample Type: Cell Culture Supernates
    5. miR-125b develops chemoresistance in Ewing sarcoma/primitive neuroectodermal tumor.
      Authors: Iida, Keiichir, Fukushi, Jun-Ichi, Matsumoto, Yoshihir, Oda, Yoshinao, Takahashi, Yusuke, Fujiwara, Toshifum, Fujiwara-Okada, Yuko, Hatano, Mihoko, Nabashima, Akira, Kamura, Satoshi, Iwamoto, Yukihide
      Cancer Cell Int, 2013;13(1):21.
      Species: Human
      Sample Type: Cell Culture Supernates
    6. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect.
      Authors: Ghezali L, Leger D, Limami Y, Cook-Moreau J, Beneytout J, Liagre B
      Exp Cell Res, 2013;319(7):1043-53.
      Species: Human
      Sample Type: Cell Lysates
    7. Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.
      Authors: Bai X, Feldman N, Chmura K, Ovrutsky A, Su W, Griffin L, Pyeon D, McGibney M, Strand M, Numata M, Murakami S, Gaido L, Honda J, Kinney W, Oberley-Deegan R, Voelker D, Ordway D, Chan E
      PLoS ONE, 2013;8(4):e61925.
      Species: Human
      Sample Type: Cell Culture Supernates
    8. A systems approach identifies HIPK2 as a key regulator of kidney fibrosis.
      Authors: Jin Y, Ratnam K, Chuang PY, Fan Y, Zhong Y, Dai Y, Mazloom AR, Chen EY, D'Agati V, Xiong H, Ross MJ, Chen N, Ma'ayan A, He JC
      Nat. Med., 2012;18(4):580-8.
      Species: Human
      Sample Type: Cell Lysates
    9. Therapeutic effect of Aralia cordata extracts on cartilage protection in collagenase-induced inflammatory arthritis rabbit model.
      Authors: Park DS, Huh JE, Baek YH
      J Ethnopharmacol, 2009;125(2):207-17.
      Species: Rabbit
      Sample Type: Serum
    10. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling.
      Authors: Granata R, Settanni F, Biancone L, Trovato L, Nano R, Bertuzzi F, Destefanis S, Annunziata M, Martinetti M, Catapano F, Ghè C, Isgaard J, Papotti M, Ghigo E, Muccioli G
      Endocrinology, 2007;148(2):512-29.
      Species: Hamster
      Sample Type: Cell Lysates
    11. Bakuchiol-induced caspase-3-dependent apoptosis occurs through c-Jun NH2-terminal kinase-mediated mitochondrial translocation of Bax in rat liver myofibroblasts.
      Authors: Park EJ, Zhao YZ, Kim YC, Sohn DH
      Eur. J. Pharmacol., 2007;559(2):115-23.
      Species: Mouse
      Sample Type: Cell Lysates
    12. Low dose leflunomide activates PI3K/Akt signalling in erythroleukemia cells and reduces apoptosis induced by anticancer agents.
      Authors: Leger DY, Liagre B, Beneytout JL
      Apoptosis, 2006;11(10):1747-60.
      Species: Human
      Sample Type: Cell Lysates
    Expand to show all 12 Citations
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