FADD antibody (AA 1-208)

Catalog No. ABIN5541516

The Mouse Monoclonal anti-FADD antibody has been validated for WB. It is suitable to detect FADD in samples from Human and Mouse.

Quick Overview for FADD antibody (AA 1-208) (ABIN5541516)

Target: FADD (Fas (TNFRSF6)-Associated Via Death Domain (FADD))

Reactivity: Human, Mouse

Host: Mouse

Clonality: Monoclonal

Conjugate: This FADD antibody is un-conjugated

Application: Western Blotting (WB)

Clone: 1F7

Product Details anti-FADD Antibody

Binding Specificity: AA 1-208

Specificity: This antibody reacts with FADD.

Purification: Protein A agarose

Immunogen: Recombinant full-length human FADD (1-208 aa)

Isotype: IgG1

Application Details

Application Notes: Western blot: 1 μg/mL for chmiluminescence detection system. 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 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 3 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 5 minutes. Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting Jurkat, HeLa, ZR-75-1, NIH/3T3, WR19L)

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 FADD

Target: FADD (Fas (TNFRSF6)-Associated Via Death Domain (FADD))

Alternative Name: fadd

Background: FADD (Fas-associated death domain protein)/MORT1 has been identified as a protein that associates specifically with the intracellular domain of Fas. It contains death domain (D D) and death effecter domain (DED) as well. DD is found in several death-inducing receptors of the TNF family, including Fas (CD95/APO-1) and TNFR-1. DED is found at N-terminus of FADD and it also present within the IC E-like protease, caspase-8 (FLICE/Mch5/MACH) and caspase-10 (FLICE2). Both DD and DED are able to a ssociate with homologous regions in other proteins, and thus prompt binding of such proteins to one another. Upon activation Fas, it relays death signals through DD, whic h interacts with the DD of the adaptor molecules FADD, recruiting them to the membrane. FADD then associat es with caspase-8 through DED, leading to the assembly of a death-inducing signaling complex (DISC). DISC-associated caspase-8 subsequently initiates proteolytic activation of other caspase, which in turn leads to apoptosis.

UniProt: Q13158

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