CRADD antibody

Catalog No. ABIN5540081

This Mouse Monoclonal antibody specifically detects CRADD in WB. It exhibits reactivity toward Human.

Quick Overview for CRADD antibody (ABIN5540081)

Target: CRADD (CASP2 and RIPK1 Domain Containing Adaptor with Death Domain (CRADD))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This CRADD antibody is un-conjugated

Application: Western Blotting (WB)

Clone: 4B12

Product Details anti-CRADD Antibody

Specificity: This antibody reacts with RAIDD.

Cross-Reactivity (Details): Does not react with Mouse (WR19L, Ba/F3), Rat (PC12, Rat-1), and Hamster (BHK).

Purification: Protein A agarose

Immunogen: Recombinant full-length human RAIDD (200 a.a.)

Isotype: IgG1

Application Details

Application Notes: Western blot: 1 μg/mL for chemiluminescence detection system. For details see protocol below.

Protocol: SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volumes 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 an 8 mg/mL solution. 3) Mix the sample with an 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 specific 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 suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.) 8) Wash the membrane with PBS-T [0.05 % Tween-20 in PBS] (10 minutes x 3 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 (10 minutes x 3 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriate chemilum inescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 1 minute. Develop the film as usual. The conditions for exposure and development may vary. (Positive controls for Western blotting Jurkat, HeLa)

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. Contains no preservatives.

Preservative: Without preservative

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 CRADD

Target: CRADD (CASP2 and RIPK1 Domain Containing Adaptor with Death Domain (CRADD))

Alternative Name: raidd

Background: Apoptosis is a major form of cell death characterized by severa l morphological features that include chromatin condensation and fragmentation, cell membrane blebbing, and formation of apoptotic bodies. These morphological changes occur via signaling pathway that leads to the recruitment and activation of caspases, a family of cysteine-containing , aspartate-specific proteases. Adaptor molecules that contai n protein-protein interaction motifs mediate the coupling of caspases to signaling pathway. For example, in the TNF receptor I mediated apoptosis pathway, the apoptotic signal is transduced through interaction of the oligomerized receptor death domain (DD) with a set of cy toplasmic adaptor/signaling molecules including FADD, TRADD and RIP. RAIDD (also known as CRADD) is another candidate adaptor molecule. It has a bipartite architecture comprising a carboxy-terminal DD and an amino-terminal caspase recruitment domain (CARD) highly homologous with the sequence of the prodomain of human caspase-2 and C. elegans CED-3. RAIDD interacts with RIP on respective DD, and with caspase-2 or CED-3 on respective CARD. Thus it may play a role in the TNF-receptor I mediated apoptosis by recruiting caspase-2 to the pathway. However, it has been remained unclea r about the significance of caspase-2 activation through the RIP/RAIDD pathway for death induction at least in some situation, because targeted disruption of RIP in mice, as well as mutational ablation of RIP function in cultured cells did not interfere with death induction by TNF nor by Fas, while NF- κ B activation through RIP was abolished in these cells.

UniProt: P78560

Pathways: Apoptosis, Caspase Cascade in Apoptosis, Positive Regulation of Endopeptidase Activity