Phospho-PKA R2 (S99) 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 Pig | ||
| MW (kDa) | 51kDa | ||
| 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-PKA R2 (S99) Rabbit Recombinant mAb detects endogenous level of total Phospho-PKA R2 (S99). |
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| 背景 | Intracellular effects of cAMP are conveyed by cyclic AMP-dependent protein kinase (PKA) types I and II in eukaryotic cells. The PKA type II holoenzyme consists of two catalytic subunits and two regulatory subunits (RIIα or RIIβ) that bind to and inactivate the catalytic subunits. PKA is activated by the binding of two cAMP molecules to each R subunit, which promotes release of the catalytic subunits from the R-cAMP complex. Active catalytic subunits phosphorylate specific substrates and can be translocated to the nucleus, where they activate cAMP-responsive genes. The subcellular localization and activity of protein kinases is often altered by protein phosphorylation. RII can be autophosphorylated by the catalytic subunit of PKA. Human RIIα is autophosphorylated on S99 throughout the cell cycle, and is hyperphosphorylated on T54 at mitosis and by purified CDK1 in vitro. |
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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