PTEN Rabbit Recombinant mAb

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  • WB
规格 价格 库存 购买数量
20ul 447.67 现货
100ul 1500.05 现货
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400-668-6834

info@selleck.cn

 

使用信息

抗体应用 WB,ELISA
稀释比例
WB
1:1000
反应性 Human Mouse Rat
MW (kDa) 54kDa
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.

Datasheet & SDS

生物描述

特异性 PTEN Rabbit Recombinant mAb可检测PTEN的内源性水平。
背景 Phosphatase and tensin homolog deleted on chromosome ten (PTEN)是一种在癌症中常发生改变的磷酸酶。PTEN具有依赖于其磷酸酶活性的作用,也有不依赖于其磷酸酶活性的作用。PTEN的遗传学改变将导致蛋白合成、细胞周期、迁移、生长、DNA修复和生存信号的失调。PTEN的细胞定位、稳定性、构象和磷酸酶活性被一系列蛋白-蛋白间相互作用和翻译后修饰所调控。因此,PTEN相互作用、修饰的蛋白对PTEN发挥肿瘤抑制功能具有深刻的影响。PTEN是一种双重特异性蛋白和脂磷酸酶,它的主要的胞内底物是第二信使PIP3,PIP3水解成为PIP2。PTEN通过抑制PIP3依赖性过程如AKT的膜募集和激活,抑制PIP3,从而抑制细胞生存、生长和增殖。PTEN因此是抑制致癌性转化中的关键节点。PTEN也是胚胎发育所必需的蛋白。

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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