PYK2 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) | 116kDa | ||
| 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 | PYK2 Rabbit Recombinant mAb detects endogenous level of total PYK2. |
|---|---|
| 背景 | Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. PYK2 and FAK comprise the focal adhesion kinase subfamily of non-receptor tyrosine kinases. PYK2 and FAK are large multidomain proteins containing an N-terminal FERM domain, a central catalytic domain, and a C-terminal segment containing dual proline rich (PR) subdomains and a focal adhesion targeting (FAT) region. While FAK is widely expressed, PYK2 expression is relatively restricted with highest levels in brain and the hematopoeitic system. Unlike FAK, optimal PYK2 activation is dependent on Ca2+ mobilization. The catalytic activity of Pyk2 is regulated by phosphorylation. Pyk2 has four major sites of tyrosine phosphorylation, Tyr-402, Tyr-579, Tyr-580, and Tyr-881. Tyrosine 402 is an autophosphorylation site that, once phosphorylated, can bind to the SH2 domain of Src family kinases (SFK). The current model for the activation of this kinase is that upon stimulation, Pyk2 becomes autophosphorylated as a result of trans-autophosphorylation, which allows for the recruitment of SFK. SFK phosphorylate additional tyrosine residues within the protein thus enhancing catalytic activity (Tyr-579/Tyr-580) and providing a new docking site (Tyr-881) for SH2 domain-containing proteins. The focal adhesion kinases FAK and Pyk2 are uniquely situated to act as critical mediators for the activation of signaling pathways that regulate cell migration, proliferation, and survival. |
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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