Phospho-Raf1 (S621) 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 Mouse Rat | ||
| MW (kDa) | 73kDa | ||
| 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-Raf1 (S621) Rabbit Recombinant mAb detects endogenous levels of phosphorylated Raf1 (S621). |
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| 背景 | The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. The regulation of Raf-1 activity is complex, involving inter- and intramolecular protein interactions as well as direct phosphorylation. In a quiescent cell, Raf-1 exists in an inactive state in the cytosol. The inactive conformation of Raf-1 is maintained by autoinhibitory interactions occurring between the N-terminal regulatory and the C-terminal catalytic domains and by the binding of a 14-3-3 dimer that contacts two phosphorylation sites, S259 and S621. That is, Raf-1 in quiescent cells was highly phosphorylated on two sites, S259 and S621. Binding of 14-3-3 proteins to Raf-1 molecules phosphorylates on S259 and S621 is thought to maintain Raf-1 in an inactive, autoinhibited conformation. Typically, the activation of Raf-1 is initiated by its interaction with Ras, which leads to the relocalization of cytosolic Raf-1 to the plasma membrane. Ras binding also promotes conformational changes that relieve Raf-1 autoinhibition and facilitate the phoshorylation of activating sites. These activating sites are found in the Raf-1 catalytic domain and include S338, Y341, T491, and S494. |
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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