FADD (1-208) Mouse Monoclonal Antibody [Clone ID: 1F7]

CAT#: AM26589AF-N

FADD (1-208) mouse monoclonal antibody, clone 1F7, Azide Free

Datasheet
SDS

Product Images

Specifications

Product Data
Clone Name 1F7
Applications WB
Recommended Dilution Western blot: 1 µg/ml for chmiluminescence detection system.
For details see protocol below.
Reactivities Human, Mouse
Host Mouse
Isotype IgG1
Clonality Monoclonal
Immunogen Recombinant full-length human FADD (1-208 aa)
Specificity

This antibody reacts with FADD.
Formulation

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


State: Azide Free
State: Liquid Ig fraction
Concentration lot specific
Purification Protein A agarose
Conjugation Unconjugated
Storage Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing.
Stability Shelf life: One year from despatch.
Database Link
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.
Synonyms FAS-associated death domain protein, MORT1, GIG3
Note

This product was originally produced by MBL International.



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)
Reference Data

Documents

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  • Antibody and Western Standard
SDS