Caspase-8 Antibody (746109)

Caspase‑8 was detected in immersion fixed Jurkat human acute T cell leukemia cell line treated for 4 hours with staurosporin using Mouse Anti-Human Caspase‑8 Polyclonal Antibody (Catalog # MAB8135) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (yellow; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

• Reactivity: Hu
• Applications: ICC/IF
• Clone: 746109
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

Caspase-8 Antibody (746109) Summary

• Immunogen: KLH-coupled Caspase-8 synthetic peptide
  CGIPVETD
  Accession # Q14790
• Specificity: Detects human cleaved Caspase-8 in ELISA.
• Source: N/A
• Isotype: IgG1
• Clonality: Monoclonal
• Host: Mouse
• Gene: CASP8
• Purity: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 8-25 ug/mL
• Control:

  Caspase-8 Knockout HeLa Cell Lysate
  Caspase-8 Knockout HeLa Cell Lysate

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Purity: Protein A or G purified from hybridoma culture supernatant
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Caspase-8 Antibody (746109)

• AIS
• androgen receptor
• CASP8
• Caspase8
• Caspase-8
• DHTRTFM
• Dihydrotestosterone receptorHYSP1
• HUMARA
• Mch5
• NR3C4KD
• Nuclear receptor subfamily 3 group C member 4
• SMAX1SBMA
• spinal and bulbar muscular atrophy

Background

Caspase-8 (Cysteine-aspartic acid protease 8/Casp8a; also named MCH5, FLICA and MACH alpha 1) is a 28 kDa member of the peptidase C14A family of enzymes (1, 2, 3). It is widely expressed and is considered an initiating caspase for the apoptotic cascade (4). Caspase-8 acts on a wide variety of substrates, including procaspases-3, 4, 6, 7, 9 and 10, c-FLIP_L and procaspase‑8 itself (1, 5, 6). Human procaspase-8a is a 54‑56 kDa, 479 amino acid (aa) protein (4, 7, 8, 9). It contains two N-terminal death domains (aa 1‑177), followed by a catalytic site that utilizes His317Gly318 plus Cys360. Normally, it is an inactive, cytosolic monomer (1, 10, 11). But following death-domain (DD) containing receptor oligomerization, Caspase-8 is recruited to the death-inducing signaling complex (DISC) that forms around the death domains of the oligomerized receptor (12). FADD/CAP-1 is recruited first, followed by procaspase-8/CAP-4 and, possibly, c-FLIP_L and procaspase‑10 (12). The recruitment, or concentration, of procaspase-8 induces homodimerization. This act alone is sufficient for activation. However, the activity level is modest at best, and appears to be directed towards either itself, or c-FLIP_L, which is known to form a functional heterodimer with procaspase-8 (5, 11). When directed towards itself, autocleavage occurs first between Asp374Ser375, generating a 43 kDa (p43) N-terminal (aa 1‑374) and an 11 kDa C‑terminal (aa 375‑479) fragment. The C‑terminus is further cleaved between Asp384Leu385 to generate a mature p10 subunit (aa 385‑479). The p43 subunit is next cleaved twice, once between Asp216Ser217, and again between Asp210Ser211 to generate a 26 kDa DD-containing prodomain (aa 1‑210) with an additional 18 kDa mature p18 subunit (aa 217‑374) (12). p18 and p10 noncovalently associate to form a 28 kDa heterodimer, which subsequently associates with another p18:p10 heterodimer to form an active, mature Caspase-8 molecule. This leaves the DISC to act on downstream apoptotic procaspases. In the event procaspase-8 comes to the DISC complexed with c‑FLIP_L, c‑FLIP_L will be cleaved by procaspase-8, generating a p43 fragment that is analogous to the Caspase-8 p43 subunit. This fragment, however, appears not to be an intermediate in a proteolytic cascade. Rather, it serves as a functional subunit, interacting with TRAF2 and activating NF kappa B. This may account for many of the nonapoptotic activities associated with Caspase-8 (5, 6, 13). Mature human and mouse Caspase-8a heterodimers are 73% aa identical (14).

1. Chowdhury, I. et al. (2008) Comp. Biochem. Physiol. B 151:10.
2. Boatright, K.M. & G.S. Salvesen (2003) Curr. Opin. Cell Biol. 15:725.
3. Launay, S. et al. (2005) Oncogene 24:5137.
4. Srinivasula, S.M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:14486.
5. Hughes, M.A. et al. (2009) Mol. Cell 35:265.
6. Lamkanfi, M. et al. (2007) Cell Death Differ. 14:44.
7. Fernandes-Alnemri, T. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7464.
8. Boldin, M.P. et al. (1996) Cell 85:803.
9. Muzio, M. et al. (1996) Cell 85:817.
10. Donepudi, M. et al. (2003) Mol. Cell 11:543.
11. Boatright, K.M. et al. (2003) Mol. Cell 11:529.
12. Golks, A. et al. (2006) Cell Death Differ. 13:489.
13. Scaffidi, C. et al. (1997) J. Biol. Chem. 272:26953.
14. Sakamaki, K. et al. (1998) Eur. J. Biochem. 253:399.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

FAQs for Caspase-8 Antibody (MAB8135). (Showing 1 - 1 of 1 FAQ).

1. May we ask the MW difference between the active form and the proform of Caspase 8 while performing WB?
   • In treated cells induced to undergo apoptosis, caspase-8 migrates as a 55/53 kDa (pro-form), 41/42 kDa (a cleaved/active or intermediate form), and 18 kDa (active form). The proform (55/53 kDa) is still seen in treated cells because not all cells undergo apoptosis at once.

Bioinformatics

• Gene Symbol: CASP8
• Uniprot:
  • Q14790()