Caspase 8 antibody (C-Term)

Catalog No. ABIN5541755

This Mouse Monoclonal antibody specifically detects Caspase 8 in WB and IP. It exhibits reactivity toward Human.

Quick Overview for Caspase 8 antibody (C-Term) (ABIN5541755)

Target: Caspase 8 (CASP8)

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This Caspase 8 antibody is un-conjugated

Application: Western Blotting (WB), Immunoprecipitation (IP)

Clone: 5D3

Product Details anti-Caspase 8 Antibody

Binding Specificity: AA 180-480, C-Term

Specificity: This antibody reacts with caspase-8.

Cross-Reactivity (Details): Does not work with Mouse (WR19L) and Rat (PC12).

Purification: Protein A agarose

Immunogen: Recombinant human caspase s-8 corresponding to C-terminal amino acids (180-480 aa)

Isotype: IgG2b

Application Details

Application Notes: Western blot: 1 μg/mL for chemiluminescence detection system. Immunoprecipitation: 2 μg/500 μL of cell extract. For details see protocol below.

Protocol: SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the cold Lysis buffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli's sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20 % MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1 % skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.) 8) Wash the membrane with PBS-T [0.05 % Tween-20 in PBS] (5 minutes x 6 times). 9) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG diluted with 1 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS-T (5 minutes x 6 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriate chemilumin escence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 3 minutes. Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting Jurkat, Raji, HeLa, U937, MCF7, HEp-G2, ZR-75-1) Immunoprecipitation 1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. 3) Add primary antibody as suggest in the APPLICATIONS into 500 μ L of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4 o C. Add 20 μ L of 50 % protein A agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4 o C. 4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds). 5) Resuspend the beads in 20 μ L of Laemmli's sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 μ L/lane for the SDS-PAGE analysis. (See SDS-PAGE & Western blotting.)

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. No preservative is contained.

Preservative: Azide free

Storage: -20 °C

Storage Comment: Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Target Details for Caspase 8

Target: Caspase 8 (CASP8)

Alternative Name: caspase-8,flice

Background: Caspase-8 (FLICE/MACH/Mch5) is a member of the ICE (interleukin- 1 converting enzyme)/CED-3 family cysteine protease. It is the most upstream protease that recei ves the activation signal from the Fas (APO1/CD95) and TNFR1 (Tumor Necrosis Factor Receptor 1) to initiate the ap optotic protease cascade that leads to activation of ICE/CED-3 family proteases. Caspase-8 has high homologous region to the ICE/CED-3 family in C-terminal and two death effecter domains (DED) in N-terminal. Binding of caspase-8 to FADD (MORT1) through association of their DED, and consequent activation of the caspases by th eir proteolytic cleavage, are thought to be critical steps in the initiation of Fas- and TNFR1-induced apoptosis. Recen tly the inhibitor of Fas- and TNFR1-induced apoptosis is identified, called I-FLICE (FLIP/Casper/FLAME/CASH). I-FLICE has high homology to caspase-8 and it contains two DED, which interacts with caspase-8 and FADD, and potently inhibits Fas- and TNFR1-induced apoptosis.

UniProt: Q14790

Pathways: Apoptosis, Caspase Cascade in Apoptosis, TLR Signaling, Activation of Innate immune Response, Tube Formation, Positive Regulation of Endopeptidase Activity, Toll-Like Receptors Cascades