IRF7 Rabbit Recombinant mAb
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使用信息
| 抗体应用 | WB,ELISA | ||
|---|---|---|---|
| 稀释比例 |
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| 反应性 | Human Mouse Rat | ||
| MW (kDa) | 54kDa | ||
| 抗体类型 | Rabbit | ||
| 浓度 | 1mg/ml | ||
| 储存液配方 | 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. | ||
| 储存条件 (自收到货起) |
Store at –20°C. |
生物描述
| 特异性 | IRF7 Rabbit Recombinant mAb detects endogenous levels of total IRF7. |
|---|---|
| 背景 | IRF7 is a multifunctional transcription factor originally identified in the context of Epstein-Barr virus (EBV) infection, and has since emerged as the crucial regulator of type I interferons (IFNs) against pathogenic infections, which activate IRF7 by triggering signaling cascades from pathogen recognition receptors (PRRs) that recognize pathogenic nucleic acids. Activation of IRF7 is prerequisite for its functions as a transcription factor. Inactive IRF7 resides in the cytoplasm as a "latent" form. Pathogenic infection triggers IRF7 phosphorylation and translocation into the nucleus, where with other co-activators it forms a transcriptional complex that binds to the promoter regions of target genes to activate transcription. IRF7 is activated by pathogenic nuclei acids through pathways mediated by TLR3, −7 and −9, RIG-I and likely DNA-dependent activator of IRF and IFI16, as well as by TLR2-mediated signaling pathway. It is a lymphoid-specific factor, which is constitutively expressed in the cytoplasm in B cells, pDCs and monocytes in the spleen, thymus, and peripheral blood lymphocytes, and is potently inducible by type I IFNs, virus infection and other stimuli such as 12-o-tetradecanoylphonol-13-acetate, TNFα and lipopolysaccharide in various cell types. IRF7 remains at low levels in most cell types, and has a very short half-life of 0.5-1 h in infected cells and approximately 5 h in uninfected cells but can be stabilized by the 26S proteasome inhibitor, MG132, or by a dominant-negative mutant of Cul1, a component of the SKP1-CUL1-F-box E3 ubiquitin ligase complex, suggesting that its stability is controlled by the ubiquitin–proteasome system. IRF7 can be regulated by various posttranslational modifications, including phosphorylation, ubiquitination, sumoylation and acetylation. It functions as the "master" regulator of type I IFN production, to be involved in the regulation of oncogenesis and apoptosis, implicated in autoimmune diseases and so on. |
实验方法
| WB |
Western Blotting Sample preparation 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 a. Blocking 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. |
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