FKBP12 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) | 12kDa | ||
| 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 | FKBP12 Rabbit Recombinant mAb detects endogenous levels of total Mutant FKBP12. |
|---|---|
| 背景 | FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor β (TGF-β) type I receptor. FKBP12 occurs in all tissues, with particularly high densities in the brain, and appears to have diverse functions. It is a subunit of two intracellular calcium release channels, the inositol 1,4,5-trisphosphate receptor and the ryanodine receptor, and is also a subunit of the TGF-β type I receptor. FKBP12 inhibits basal signaling of these three receptors and also regulates the P-glycoprotein multidrug transporter. Binding of FKBP12 to FK506 and calcineurin forms a ternary complex to inhibit the serine/threonine phosphatase activity of calcineurin, which is important for several cellular processes such as T-cell activation. In addition, FKBP12 exhibits many other functions which involve binding to different cellular receptors or targets. For example, in the absence of FK506, FKBP12 binds to the ryanodine receptor, which is one of the major calcium-release channels in the sarcoplasmic and endoplasmic reticula. Interaction between FKBP12 and ryanodine receptor stabilizes the ryanodine receptor channel and modulates channel gating, leading to increased channel conductance levels and mean open-time. FKBP12 has also been shown to interact with transforming growth factor-β type I receptor to inhibit receptor-mediated signal transduction. Furthermore, FKBP12 has an inhibitory effect on the cellular activity of epidermal growth factor receptor by modulating the receptor's phosphorylation status. |
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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