DAXX antibody (C-Term)

Catalog No. ABIN501203

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

Quick Overview for DAXX antibody (C-Term) (ABIN501203)

Target: DAXX (Death-Domain Associated Protein (DAXX))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This DAXX antibody is un-conjugated

Application: Western Blotting (WB), Immunofluorescence (IF)

Clone: DAXX-01

Product Details anti-DAXX Antibody

Binding Specificity: AA 558-740, C-Term

Specificity: This antibody reacts with Daxx (110 kDa).

Cross-Reactivity (Details): Species reactivity (tested):Human.

Purification: Protein-A Sepharose Chromatography of hybridoma supernatant.

Immunogen: Recombinant C-terminal part (aa 558-740) of Human Daxx

Isotype: IgG1

Application Details

Application Notes: Western blotting: 1 μg/mL for chemiluminescence detection system. Immunocytochemistry: 10 μg/mL. Detailed procedure is provided in Protocols.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Protocol: SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mMTris-HCl, pH 7. 2, 250 mM NaCl, 0. 1% NP-40, 2 mM EDTA, 10%glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. Measure the protein concentration of the supernatant and add the cold Lysisbuffer 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 μL of the sample per lane in a 1mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hourin 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, pH7. 2) for 1 hour at room temperature, or overnight at 4°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. (Theconcentration of antibody will depend on condition. )8) Wash the membrane with PBS-T [0. 05% Tween-20 in PBS] (5 minutes x 3 times). 9) Incubate the membrane with the 1: 10,000 HRP-conjugated anti-mouse IgG diluted with1% skimmed milk (in PBS, pH 7. 2) for 1 hour at RT. 10) Wash the membrane with PBS-T (10 minutes x 3 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriatechemiluminescence reagent for 1 minute. 12) Remove extra reagent from the membrane by dabbing with paper towel, and seal it inplastic wrap. 13) Expose to an X-ray film in a dark room for 3 minutes. 14) Develop the film as usual. The condition for exposure and development may vary. Positive Controls: Jurkat, Raji. Immunocytochemistry1) Culture the cells in the appropriate condition on a glass slide. (for example, spread 10e4of HEp-II cells for one slide, then incubate in a CO2 incubator for one night. )2) Fix the cells by immersing the slide in MeOH containing methanol for 10 minutes on ice. 3) Air dry the slides. 4) Add the primary antibody diluted with PBS as suggest in the APPLICATIONS onto thecells and incubate for 1 hour at room temperature (Optimization of antibody concentrationor incubation condition are recommended if necessary). 5) Prepare a wash container such as a 500 mL beaker with a stirrer. Then wash the culturedcells on the glass slide by soaking the slide with a plenty of PBS in the wash container for 5minutes. Take care not to touch the cells. Repeat another washes once more. 6) Add the secondary antibody 1: 100 FITC conjugated anti-mouse IgG onto the cells. Incubate for 1 hour at room temperature. Keep out light by aluminum foil. 7) Wash the slide in a plenty of PBS as in the step 5). 8) Wipe excess liquid from slide but take care not to touch the cells. Never leave the cellsto dry. 9) Promptly add PermafluorTM aqueous mounting medium onto the slide, then put a cover

Restrictions: For Research Use only

Handling

Concentration: 1.0 mg/mL

Buffer: PBS, pH 7.2 containing 50 % Glycerol without preservatives.

Preservative: Without preservative

Handling Advice: Avoid repeated freezing and thawing.

Storage: -20 °C/-80 °C

Storage Comment: Store lyophilized (preferably in a desiccator) at -20 °C and in aliquots at -80 °C. Reconstituted antibody can be stored at 4 °C for a limited period of time, it does not show decline in activity after two weeks at 4 °C.

Target Details for DAXX

Target: DAXX (Death-Domain Associated Protein (DAXX))

Alternative Name: DAXX

Background: Daxx binds specifically to the cytosolic Death Domain (DD) of the Fas receptor and enhances Fas-induced apoptosis. Daxx also links this receptor to an apoptosis pathway involving activation of c-Jun N-terminal kinase (JNK). Daxx is widely expressed in fetal and adult tissues, indicating its important function in Fas signaling pathways. The Daxx protein resides primarily in the nucleus but it is sequestered in the cytoplasm by ASK1 (apoptosis signal-regulating kinase 1), resulting in inhibition of Daxx-mediated transcriptional regulation.Synonyms: BING2, DAP6, Death domain-associated protein 6, EAP1, ETS1-associated protein 1, Fas death domain-associated protein

Gene ID: 1616

NCBI Accession: NP_001135441

UniProt: Q9UER7

Pathways: Intracellular Steroid Hormone Receptor Signaling Pathway