Caspase-8 Antibody [Unconjugated]

Western blot shows lysates of Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) with 1 mM staurosporine (STS) for for 3 hours. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human/Mouse Caspase‑8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1650), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). For additional reference Recombinant Human Caspase-8 (Catalog # 705‑C8) was included. Specific bands were detected for Caspase‑8 precursor at approximately 57-60 kDa (as indicated). In STS-treated samples, specific bands were detected for Caspase-8 p41/43 subunit at approximately 41 and 43 kDa (as indicated) and Caspase-8 p18, p14, and p10 subunits at approximately 18 kDa, 14 kDa, and 10 kDa, respectively (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 4.

Simple Western lane view shows lysates of Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) with 1 mM Staurosporine (STS) for 3 hours, loaded at 0.2 mg/mL. Specific bands were detected for Caspase‑8 at approximately 58-62 kDa (as indicated) using 5 µg/mL of Rabbit Anti-Human/Mouse Caspase‑8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1650). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

• Reactivity: Hu, Mu
• Applications: WB, Simple Western
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Concentration: LYOPH

Caspase-8 Antibody [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human Caspase-8
  Ser217-Asp384 (Asp285His) (p18 subunit), Leu385-Asp479 (p10 subunit)
  Accession # Q14790
• Specificity: Detects human/mouse Caspase-8 and cleavage products. Detects multiple isoforms of Caspase-8.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: CASP8

Applications/Dilutions

• Dilutions:
  • Western Blot 0.5 ug/mL
  • Simple Western 5 ug/mL
• Application Notes: In Simple Western only 10-15 uL of the recommended dilution is used per data point.
• Control:

  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
• Reconstitution Instructions: Reconstitute at 0.2 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 [Unconjugated]

• 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.

Publications for Caspase-8 Antibody (AF1650)(7)

Publications using AF1650

• M Ijiri, M Fujiya, H Konishi, H Tanaka, N Ueno, S Kashima, K Moriichi, J Sasajima, K Ikuta, T Okumura Ferrichrome identified from Lactobacillus casei ATCC334 induces apoptosis through its iron-binding site in gastric cancer cells Tumour Biol., 2017;39(6):1010428317711. 2017 [PMID: 28639907] (Western Blot, Xenograft)
• Masoumi-Moghaddam, Samar, Amini, Afshin, Ehteda, Anahid, Wei, Ai-Qun, Morris, David La The expression of the Sprouty 1 protein inversely correlates with growth, proliferation, migration and invasion of ovarian cancer cells. J Ovarian Res, 2104;7(0):61. 2104 [PMID: 24932220] (Western Blot, Human)
• Lu, Jiahong, He, Liqiang, Behrends, Christia, Araki, Masatake, Araki, Kimi, Jun Wang, Qing, Catanzaro, Joseph M, Friedman, Scott L, Zong, Wei-Xing, Fiel, M Isabel, Li, Min, Yue, Zhenyu NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity. Nat Commun, 2014;5(0):3920. 2014 [PMID: 24849286] (IHC, Mouse)
• Cha Y, Kim D, Hyun J, Kim S, Park K TCEA3 binds to TGF-beta receptor I and induces Smad-independent, JNK-dependent apoptosis in ovarian cancer cells. Cell Signal, 2013;25(5):1245-51. 2013 [PMID: 23357533] (Western Blot, Human)
• Pan, Ji-An, Fan, Yongjun, Gandhirajan, Rajesh K, Madesh, Muniswam, Zong, Wei-Xing Hyperactivation of the mammalian degenerin MDEG promotes caspase-8 activation and apoptosis. J Biol Chem, 2013;288(5):2952-63. 2013 [PMID: 23239879] (Western Blot, Mouse)
• Pan JA, Ullman E, Dou Z Inhibition of protein degradation induces apoptosis through a microtubule-associated protein 1 light chain 3-mediated activation of caspase-8 at intracellular membranes. Mol. Cell. Biol., 2011;31(15):3158-70. 2011 [PMID: 21628531] (Western Blot, Human)
• Son KN, Becker RP, Kallio P, Lipton HL Theiler's virus-induced intrinsic apoptosis in M1-D macrophages is Bax mediated and restricts virus infectivity: a mechanism for persistence of a cytolytic virus. J. Virol., 2008;82(9):4502-10. 2008 [PMID: 18287228] (Neutralization, Mouse)

FAQs for Caspase-8 Antibody (AF1650). (Showing 1 - 1 of 1 FAQs).

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()