SignalSilence? B-Raf siRNA I

• 产品编号：CST-8935S      品牌：CST       原厂货号：8935S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

B-Raf siRNA I可以抑制人和猴的B-Raf表达。来自Cell Signaling Technology (CST)的SignalSilence^® B-Raf siRNA I可以帮助研究者通过RNA干扰特异性地抑制B-Raf的表达，这种方法可以通过将双链RNA分子传递到细胞内从而使基因表达有选择的沉默。来自CST的所有的SignalSilence^® siRNA产品都是经过内部严格检测的，并且通过Western blot 分析证明确实能够减少目的蛋白的表达。通过三苯甲基分析每个碱基以监测寡核苷酸的合成，确保合适的配对效率。随后寡核苷酸通过亲和固相萃取法纯化。退火的RNA双链通过质谱分析来证实其精确的组成。每一批产品都通过质谱分析与前面的产品进行比较，来保证不同批次之间的最大一致性。CST建议转染100 nM SignalSilence® B-Raf siRNA I，48-72小时后裂解细胞。转染的步骤请参阅转染试剂的说明书。关于使用问题，可以随时咨询CST。 每管试剂能进行100次转染，该转染次数以总体积为300μl 的1个24孔板孔中siRNA终浓度100 nM来进行计算。A-Raf, B-Raf, c-Raf (Raf-1)是由GTP结合蛋白-Ras蛋白所募集的主要效应因子，能激活MEK-MAPK途径(1)。目前对c-Raf的活化机制了解的最清楚，它涉及到许多活化位点的磷酸化，包括Ser338,Tyr341,Thr491,Ser494,Ser497和Ser499位点(2)。p21活化的蛋白激酶(PAK)能磷酸化c-Raf蛋白的Ser338位点 ，Src家族蛋白能磷酸化c-Raf蛋白的Tyr341位点，进而诱导c-Raf的活化(3,4)。c-Raf蛋白的Ser338位点，与A-Raf 的Ser299位点 、 B-Raf 的Ser445位点相对应，尽管B-Raf蛋白的Ser445位点已经被组成性的磷酸化 (5)。Akt和AMPK可以分别磷酸化c-Raf蛋白的Ser259和Ser621位点，从而抑制14-3-3与c-Raf的结合(6,7)。虽然A-Raf, B-Raf, c-Raf的序列和功能相似，但是调节方式各不相同(8)。值得关注的是，B-Raf蛋白包含三个Akt的磷酸化位点(Ser364,Ser428和Thr439位点)，但缺少与c-Raf蛋白的Tyr341位点相对应的磷酸化位点(8,9)。研究表明，B-Raf蛋白的突变体V600E，会导致激酶活性的调高，而且这种突变在恶性黑色素瘤中普遍存在(10)。c-Raf蛋白的六个位点 (Ser29, Ser43, Ser289, Ser296, Ser301和Ser642位点)会以某种方式过度磷酸化，这与c-Raf蛋白的失活有关。这六个位点的过度磷酸化依赖于下游的MEK信号转导，并使c-Raf不应答后续的活化事件(11)。

1. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
2. Chong, H. et al. (2001) EMBO J 20, 3716-27.
3. King, A.J. et al. (1998) Nature 396, 180-3.
4. Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
5. Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
7. Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
8. Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
9. Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
10. Davies, H. et al. (2002) Nature 417, 949-54.
11. Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.

原厂资料：

Homology

Species predicted to react based on 100% sequence homology: Monkey

Specificity / Sensitivity

B-Raf siRNA I inhibits human and monkey B-Raf expression.

Description

SignalSilence® B-Raf siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit B-Raf expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM SignalSilence® B-Raf siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use. 
 Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 µl per well.

Background

A-Raf, B-Raf, and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway (1). Activation of c-Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338, Tyr341, Thr491, Ser494, Ser497, and Ser499 (2). p21-activated protein kinase (PAK) has been shown to phosphorylate c-Raf at Ser338 and the Src family phosphorylates Tyr341 to induce c-Raf activity (3,4). Ser338 of c-Raf corresponds to similar sites in A-Raf (Ser299) and B-Raf (Ser445), although this site is constitutively phosphorylated in B-Raf (5). Inhibitory 14-3-3 binding sites on c-Raf (Ser259 and Ser621) can be phosphorylated by Akt and AMPK, respectively (6,7). While A-Raf, B-Raf, and c-Raf are similar in sequence and function, differential regulation has been observed (8). Of particular interest, B-Raf contains three consensus Akt phosphorylation sites (Ser364, Ser428, and Thr439) and lacks a site equivalent to Tyr341 of c-Raf (8,9). Research studies have shown that the B-Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma (10). Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301, and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation. The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events (11).

1. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
2. Chong, H. et al. (2001) EMBO J 20, 3716-27.
3. King, A.J. et al. (1998) Nature 396, 180-3.
4. Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
5. Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
7. Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
8. Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
9. Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
10. Davies, H. et al. (2002) Nature 417, 949-54.
11. Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.

注意事项：

Storage: B-Raf siRNA I is supplied in RNAse-free water. Aliquot
and store at -20ºC