Overview

  • Product nameCaspase 8 Assay Kit (Colorimetric)
    See all Caspase-8 kits
  • Detection methodColorimetric
  • Sample type
    Cell Lysate
  • Assay typeEnzyme 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.



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  • Notes

    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.

  • Tested applicationsSuitable for: Functional Studiesmore details

Properties

  • FunctionMost 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 specificityIsoform 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 diseaseDefects 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 similaritiesBelongs to the peptidase C14A family.
    Contains 2 DED (death effector) domains.
  • DomainIsoform 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 localizationCytoplasm.
  • Information by UniProt
  • Alternative names
    • ALPS2B
    • Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 12 protein
    • Apoptotic cysteine protease
    • Apoptotic protease Mch-5
    • Apoptotic protease Mch5
    • CAP4
    • CASP-8
    • CASP8
    • CASP8_HUMAN
    • Caspase 8
    • Caspase 8 apoptosis related cysteine peptidase
    • Caspase-8 subunit p10
    • CED 3
    • 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
    • MCH5
    • MGC78473
    • MORT1 associated ced 3 homolog
    • MORT1-associated CED-3 homolog
    • OTTHUMP00000163717
    • OTTHUMP00000163720
    • OTTHUMP00000163724
    • OTTHUMP00000163725
    • OTTHUMP00000165062
    • OTTHUMP00000165063
    • OTTHUMP00000165064
    • OTTHUMP00000206552
    • OTTHUMP00000206582
    see all

Applications

Our Abpromise guarantee covers the use of ab39700 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Functional Studies Use at an assay dependent concentration.

Caspase 8 Assay Kit (Colorimetric) images

  • 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.

  • 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

References for Caspase 8 Assay Kit (Colorimetric) (ab39700)

This product has been referenced in:
  • Ma S  et al. Ferroptosis is induced following siramesine and lapatinib treatment of breast cancer cells. Cell Death Dis 7:e2307 (2016). Functional Studies . Read more (PubMed: 27441659) »

See 1 Publication for this product

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