|Reactivity||Human Mouse Rat|
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
Specimen Preparation (forcultured cell lines, IF-IC)
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
1. Add theblocking buffer and incubate for 60 min at RT.
|Specificity||Daxx Rabbit Recombinant mAb detects endogenous levels of total Daxx.|
|Background||The death-domain-associated protein (Daxx) could be associated with both nuclear and cytoplasmic events during apoptosis. Daxx was originally identified as a protein that specifically binds to the death domain of the transmembrane death receptor FAS (also called CD95) in the cytoplasm and potentiates FAS-induced apoptosi. Daxx is ubiquitously expressed throughout the body with particular high expression in the thymus and testes. At the cellular level, Daxx is mainly a nuclear protein that associates with several different subnuclear structures, although it also interacts with FAS at the plasma membrane. Daxx interacts with pro-apoptotic receptors such as FAS and TGFb receptor II. Daxx can activate both JNK and p38, interact with and activate the upstream JNK kinase kinase ASK1 not onlyin response to FAS stimulation, but also upon glucose deprivation. Daxx is known to repress several transcription factors, including Pax3, ETS1, E2F1, NF-kB, p53 and p73. Moreover, Daxx interacts with and inhibits Smad4 upon TGFb stimulation. Daxx was reported to interact with several crucial proteins involved in transcriptional silencing, namely histone deacetylase II, core histones and the chromatin-associated protein DEK.|