Phospho-EGFR (Y1173) 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 | ||
| MW (kDa) | 175kDa | ||
| 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-EGFR (Y1173) Rabbit Recombinant mAb detects endogenous levels of phosphorylated EGFR (Y1173). |
|---|---|
| 背景 | Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays key roles in cellular migration, proliferation, and differentiation. The six autophosphorylation sites in the COOH-terminal tail of EGFR are tyrosines 992, 1045, 1068, 1086, 1148, and 1173. Once phosphorylated, these residues serve as docking sites for intracellular signaling proteins containing Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains and provide a connection between the external stimulation and specific internal signal transduction pathways. Phospho-Y992 and -Y1173 recruit and bind to the SH2 domain of phospholipase (PL) Cγ1, resulting in phosphorylation of PLCγ1 on tyrosines 771 and 1254 by EGFR and increased phospholipase activity, which is required for EGF-stimulated cell motility. when phosphorylated, Y1173 serves as a docking site for the adaptor protein Shc, which also increases activity of the MAPK/ERK cascade. Tyr-1173 has also been demonstrated to be binding sites for phosphatases, which play an important role in Tyr dephosphorylation and hence modulation of EGFR activity. |
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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