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    caspase-8-assay-kit-colorimetric-ab39700.pdf

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Cell Biology Apoptosis Intracellular Caspases etc Caspases
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Caspase-8 Assay Kit (Colorimetric) (ab39700)

  • Datasheet
  • SDS
  • Protocol Booklet
Submit a review Q&A (1)References (16)

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Functional assays: Caspase 8 Assay Kit (Colorimetric) (ab39700)
  • Functional assays: Caspase 8 Assay Kit (Colorimetric) (ab39700)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 2 hr
  • Sample type: Cell Lysate

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Overview

  • Product name

    Caspase-8 Assay Kit (Colorimetric)
    See all Caspase-8 kits
  • Detection method

    Colorimetric
  • Sample type

    Cell Lysate
  • Assay type

    Enzyme activity
  • Assay time

    2h 00m
  • Product overview

    Caspase 8 Assay Kit (colorimetric) (ab39700) is a simple and convenient assay to quantify the activity of caspase 8 in cell lysates, based on the recognition of the sequence Ile-Glu-Thr-Asp (IETD). The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after it is cleaved from the labeled substrate IETD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at OD 400 – 405 nm. Comparison of the absorbance of pNA from an apoptotic sample with an un-induced control allows determination of the fold increase in Caspase 8 activity.



    Visit our FAQs page for tips and troubleshooting.

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K113 Caspase-8 Colorimetric Assay Kit. K113-100 is the same size as the 100 test size of ab39700.

    The caspase family of highly conserved cysteine proteases play an essential role in programmed cell death (including apoptosis, pyropoptosis and necroptosis).

    Mammalian caspases can be subdivided into three functional groups: initiator caspases (Caspase 2, 8, 9 and 10), executioner caspases (Caspase 3, 6 and 7), and inflammatory caspases (Caspase 1, 4, 5, 11 and 12). Initially synthesized as inactive pro-caspases, caspases become rapidly cleaved and activated in response to granzyme B, death receptors and apoptosome stimuli. Caspases will then cleave a range of substrates, including downstream caspases, nuclear proteins, plasma membrane proteins and mitochondrial proteins, ultimately leading to cell death.

    Caspase 8 (CASP8/FLICE, EC:3.4.22.61) is the most upstream caspase involved in the activation of apoptosis through the extrinsic pathway, mediated by CD95 (Fas) receptor and TNFR. Caspase 8 is recruited to the receptors through the adapter molecule FADD, resulting in the formation of the aggregate called death-inducing signaling complex (DISC) and proteolytic activation of caspase 8. The active dimeric enzyme is then liberated from the DISC and free to activate downstream apoptotic proteases. Inhibition or inactivation of caspase 8 is required for induction of necroptosis.

    Other caspase and apoptosis assays

    Review the full set of caspase assays, or the apoptosis assay and apoptosis marker guide.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    2X Reaction Buffer 4 x 2ml
    Cell Lysis Buffer 1 x 100ml
    Dilution Buffer 1 x 100ml
    DTT 1 x 400µl
    IETD-pNA 1 x 500µl
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Caspases etc
    • Caspases
    • Cancer
    • Invasion/microenvironment
    • Apoptosis
    • Caspases
    • Cell Biology
    • Proteolysis / Ubiquitin
    • Proteolytic enzymes
    • Other proteases
    • Kits/ Lysates/ Other
    • Kits
    • Apoptosis Kits
    • Caspase Assays Kits
    • Caspase 8 assay kits
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Apoptosis
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • Caspases
    • Cancer
    • Cell Death
    • Necroptosis
    • Cancer
    • Cell Death
    • Apoptosis
    • Metabolism
  • Function

    Most upstream protease of the activation cascade of caspases responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced cell death. Binding to the adapter molecule FADD recruits it to either receptor. The resulting aggregate called death-inducing signaling complex (DISC) performs CASP8 proteolytic activation. The active dimeric enzyme is then liberated from the DISC and free to activate downstream apoptotic proteases. Proteolytic fragments of the N-terminal propeptide (termed CAP3, CAP5 and CAP6) are likely retained in the DISC. Cleaves and activates CASP3, CASP4, CASP6, CASP7, CASP9 and CASP10. May participate in the GZMB apoptotic pathways. Cleaves ADPRT. Hydrolyzes the small-molecule substrate, Ac-Asp-Glu-Val-Asp-
    -AMC. Likely target for the cowpox virus CRMA death inhibitory protein. Isoform 5, isoform 6, isoform 7 and isoform 8 lack the catalytic site and may interfere with the pro-apoptotic activity of the complex.
  • Tissue specificity

    Isoform 1, isoform 5 and isoform 7 are expressed in a wide variety of tissues. Highest expression in peripheral blood leukocytes, spleen, thymus and liver. Barely detectable in brain, testis and skeletal muscle.
  • Involvement in disease

    Defects in CASP8 are the cause of caspase-8 deficiency (CASP8D) [MIM:607271]. CASP8D is a disorder resembling autoimmune lymphoproliferative syndrome (ALPS). It is characterized by lymphadenopathy, splenomegaly, and defective CD95-induced apoptosis of peripheral blood lymphocytes (PBLs). It leads to defects in activation of T-lymphocytes, B-lymphocytes, and natural killer cells leading to immunodeficiency characterized by recurrent sinopulmonary and herpes simplex virus infections and poor responses to immunization.
  • Sequence similarities

    Belongs to the peptidase C14A family.
    Contains 2 DED (death effector) domains.
  • Domain

    Isoform 9 contains a N-terminal extension that is required for interaction with the BCAP31 complex.
  • Post-translational
    modifications

    Generation of the subunits requires association with the death-inducing signaling complex (DISC), whereas additional processing is likely due to the autocatalytic activity of the activated protease. GZMB and CASP10 can be involved in these processing events.
    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localization

    Cytoplasm.
  • Target information above from: UniProt accession Q14790 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • ALPS2B
    • Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 12 protein
    • Apoptosis related cysteine peptidase
    • Apoptotic cysteine protease
    • Apoptotic protease Mch-5
    • Apoptotic protease Mch5
    • CAP 4
    • CAP4
    • CASP-8
    • CASP8
    • CASP8_HUMAN
    • Caspase 8
    • Caspase 8 apoptosis related cysteine peptidase
    • Caspase IIX
    • Caspase-8 subunit p10
    • caspase8
    • CED 3
    • FADD Homologous ICE/CED3 Like Protease
    • FADD Like ICE
    • FADD-homologous ICE/CED-3-like protease
    • FADD-like ICE
    • FLICE
    • FLJ17672
    • ICE-like apoptotic protease 5
    • MACH
    • MACH alpha 1/2/3 protein
    • MACH beta 1/2/3/4 protein
    • MACH5
    • MCH 5
    • MCH5
    • MGC78473
    • MORT1 associated ced 3 homolog
    • MORT1 associated CED3 homolog
    • MORT1-associated CED-3 homolog
    • OTTHUMP00000163717
    • OTTHUMP00000163720
    • OTTHUMP00000163724
    • OTTHUMP00000163725
    • OTTHUMP00000165062
    • OTTHUMP00000165063
    • OTTHUMP00000165064
    • OTTHUMP00000206552
    • OTTHUMP00000206582
    see all

Associated products

  • Corresponding Antibody

    • Anti-Caspase-8 antibody [E6] (ab32125)
    • Anti-Caspase-8 antibody (ab4052)
  • Related Products

    • Z-IETD-FMK, Caspase-8 inhibitor (ab141382)
    • Z-VEID-FMK, caspase-6 inhibitor (ab142025)
    • Z-LEHD-FMK, caspase-9 inhibitor (ab142026)
    • Ac-IEPD-CHO, granzyme B and caspase-8 inhibitor (ab142030)
    • Q-IETD-OPh, caspase-8 inhibitor (ab142038)

Images

  • Functional assays: Caspase 8 Assay Kit (Colorimetric) (ab39700)
    Functional assays: Caspase 8 Assay Kit (Colorimetric) (ab39700)

    Active caspase 8 in control (CTRL) Jurkat cells (10e6/mL) or cells treated for five hours with 10 μg/mL Cyclohexamide (CHX) (ab120093) or four hours with 25 μg/mL Mitomycin C (MitoC) (ab120797). Background signal subtracted, duplicates; +/- SD.

  • Functional assays: Caspase 8 Assay Kit (Colorimetric) (ab39700)
    Functional assays: Caspase 8 Assay Kit (Colorimetric) (ab39700)

    Active caspase 8 in control (CTRL) Jurkat cells (10e6/mL) or in cells after four hours exposure to 50 ng/mL anti-Fas Ab (αFas) (MBL), or pretreated one hour with 50 μM Z-VAD(OMe)-FMK (ab120487) followed by four hours with αFas. Background signal subtracted, duplicates; +/- SD.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (16)

Publishing research using ab39700? Please let us know so that we can cite the reference in this datasheet.

ab39700 has been referenced in 16 publications.

  • Lulijwa R  et al. Uncoupling Thermotolerance and Growth Performance in Chinook Salmon: Blood Biochemistry and Immune Capacity. Metabolites 11:N/A (2021). PubMed: 34436488
  • Liu LH  et al. Paeonol exerts anti-tumor activity against colorectal cancer cells by inducing G0/G1 phase arrest and cell apoptosis via inhibiting the Wnt/ß-catenin signaling pathway. Int J Mol Med 46:675-684 (2020). PubMed: 32626954
  • El-Huneidi W  et al. Micromeria fruticosa Induces Cell Cycle Arrest and Apoptosis in Breast and Colorectal Cancer Cells. Pharmaceuticals (Basel) 13:N/A (2020). PubMed: 32503209
  • De AK  et al. A Natural Quinazoline Derivative from Marine Sponge Hyrtios erectus Induces Apoptosis of Breast Cancer Cells via ROS Production and Intrinsic or Extrinsic Apoptosis Pathways. Mar Drugs 17:N/A (2019). PubMed: 31771152
  • He Y  et al. The SIAH1-HIPK2-p53ser46 Damage Response Pathway is Involved in Temozolomide-Induced Glioblastoma Cell Death. Mol Cancer Res 17:1129-1141 (2019). PubMed: 30796178
View all Publications for this product

Customer reviews and Q&As

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Submit a review Submit a question

Question

can you tell me what samples you tested as a control and apoptosis induced and what the fold increase was? i would like to use these as a control to know i am using the assay correctly?

Read More

Abcam community

Verified customer

Asked on May 28 2013

Answer

Thank you for contacting us.
The link to the section on homogenizing tissue in our WB protocol is:

https://www.abcam.com/index.html?pageconfig=resource&rid=13045

This can help you to prepare your samples for the kit.


Camptothecin induced Jurkat cells as the sample and the uninduced cells as the control. Based on the conc of Camptothecin and the time of incubation we get different folds of increase of the caspases.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Abcam Scientific Support

Answered on May 28 2013

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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