Phospho-ATF2 (T71) Rabbit Recombinant mAb
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使用信息
| 抗体应用 | WB,ELISA | ||
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| 稀释比例 |
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| 反应性 | Human | ||
| MW (kDa) | 70kDa | ||
| 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 | Phospho-ATF2 (T71) Rabbit Recombinant mAb detects endogenous levels of phosphorylated ATF2 (T71). |
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| 背景 | Activating transcription factor 2 (ATF2), also known as cyclic AMP (cAMP) response element (CRE) binding protein 2 (CREB2) and CRE-BP1, is a member of the activating protein-1 (AP1) transcription factor family that regulates the expression of many genes through homo-dimerization or hetero-dimerization with other AP1 family members, such as the CREB, Fos, Maf, or Jun family transcription factors. ATF2 plays an important role in transducing extracellular signals to the nucleus to facilitate transcriptional responses to stimuli. ATF2 can be activated by many stimuli, including growth factors, ultraviolet (UV) radiation, and cytokines. ATF2 transcriptional activation is mediated by stress-activated protein kinases (SAPKs) through phosphorylation at amino acids threonine-69 and threonine-71 (T69, T71). Following phosphorylation at T69/T71, ATF2 can interact with other AP1 proteins and translocate to the nucleus to modulate the expression of hundreds of genes. Phosphorylation at Thr69 and Thr71 of ATF2 appears to enhance the intrinsic histone acetyltransferase activity of ATF2 and to regulate its degradation by the ubiquitin pathway |
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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