Caspase 8 Assay Kit (Colorimetric) (ab39700)
- Datasheet
- Specific References (4)
- Protocols
Overview
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Product nameCaspase 8 Assay Kit (Colorimetric)
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Detection methodColorimetric
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Sample typeCell Lysate
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Assay typeEnzyme activity
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Assay time2h 00m
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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.
Properties
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Storage instructionsStore at -20°C. Please refer to protocols.
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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
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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.
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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.
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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.
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Post-translational
modificationsGeneration 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
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Alternative names
- ALPS2B
- Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 12 protein
- Apoptotic cysteine protease
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Associated products
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Corresponding Antibody
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Related Products
Images
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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
References
This product has been referenced in:
- DeBoever C et al. Medical relevance of protein-truncating variants across 337,205 individuals in the UK Biobank study. Nat Commun 9:1612 (2018). Read more (PubMed: 29691392) »
- 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) »
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