Raf1 Rabbit Recombinant mAb

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  • WB
规格 价格 库存 购买数量
20ul RMB 447.88 现货
100ul RMB 1500.55 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
MW (kDa) 73kDa
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 Raf1 Rabbit Recombinant mAb可检测Raf1的内源性水平。
背景 RAF激酶家族主要作为RAS下游分子传递信号。Raf-1激酶(c-Raf)处于细胞增殖、致瘤性转化、分化和凋亡调控信号网络的中心位置,主要经由MAPK/ERK通路传递信号。Raf-1磷酸化并激活MEK,而MEK则磷酸化并激活ERK。活化的Ras与Raf-1高亲和力结合,但并不直接改变Raf-1的催化活性。Ras将Raf-1从胞质定位到质膜,发生多步骤激活过程。Raf-1的激活需要与调节性蛋白、脂质、复合体相互作用,磷酸状态改变。Ser339和Tyr341的磷酸化对Raf-1的激活来说是必需的。此外,位于激活环的Thr491和Ser393的磷酸化也是必要的,但不是激活过程的充分条件。这些位点与Ser338和Tyr34相互协作。Raf-1同样可以被磷酸化负调控。cAMP依赖性激酶PKA可抑制Raf-1,磷酸化其Ser43、Ser259和Ser621。在静息细胞中,这些位点本来就处于磷酸化状态,而在PKA的作用下,被进一步诱导磷酸化。

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.

参考文献

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