SignalSilence? Chk2 siRNA I

• 产品编号：CST-6276S      品牌：CST       原厂货号：6276S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

研究人员可以使用CST的SignalSilence® Chk2 siRNA I，采用RNA干扰（一种通过传递外源性的双链RNA分子到细胞中从而能选择性的沉默基因表达的方法）技术特异性的抑制CDK2 蛋白表达。CST所有的SignalSilence® siRNA产品都经过了严格的内部测试并采用Western分析证明其可以有效降低靶蛋白的表达。 为了保证适当的结合效率，我们采用三苯甲基分析的方法逐个碱基的监测寡核苷酸的合成。随后通过亲和固相提取纯化寡核苷酸。退火的双链RNA进一步通过质谱分析来验证双链复合体的确切成分。每一批产品和前一批都要通过质谱分析进行比较以确定批次间最大限度的一致性。CST建议使用100 nM Chk2 siRNA I转染48-72小时后裂解细胞。转染的具体步骤请参考转染试剂制造商提供的操作标准。使用过程中有任何问题请随时联系CST。 Chk2是哺乳动物中和芽殖酵母Rad53和裂殖酵母Cds1检验点激酶同源的蛋白(1-3)。Chk2的氨基末端结构域包含有一个七丝氨酸或者苏氨酸残基(Ser19, Thr26, Ser28, Ser33, Ser35, Ser50 and Thr68)组成的系列，每个系列紧跟着谷氨酸(SQ 或TQ模体)。这些地方是已知的ATM/ATR激酶磷酸化的首选作用位点(4,5)。电离辐射(IR)引起DNA损伤之后，UV照射处理或者羟基脲处理，这个区域的68位苏氨酸和其他位点被ATM/ATR磷酸化(5-7)。因此，SQ/TQ簇结构域似乎具有调节功能。第68位苏氨酸的磷酸化是后续激活步骤的先决条件，激活归因于激酶结构域激活回路中Chk2第383位和387位苏氨酸残基的自体磷酸化(8)。

1. Allen, J.B. et al. (1994) Genes Dev. 8, 2401-2415.
2. Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665.
3. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
4. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
5. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
6. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
7. Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.
8. Lee, C.H. and Chung, J.H. (2001) J. Biol. Chem. 276, 30537-30541.

原厂资料：

Description

SignalSilence® Chk2 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Chk2 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 Chk2 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. 

Background

Chk2 is the mammalian orthologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (1-3). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50 and Thr68) each followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (4,5). After DNA damage by ionizing radiation (IR), UV irradiation or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (5-7). The SQ/TQ cluster domain, therefore, seems to have a regulatory function. Phosphorylation at Thr68 is a prerequisite for the subsequent activation step, which is attributable to autophosphorylation of Chk2 on residues Thr383 and Thr387 in the activation loop of the kinase domain (8). 

1. Allen, J.B. et al. (1994) Genes Dev. 8, 2401-2415.
2. Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665.
3. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
4. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
5. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
6. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
7. Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.
8. Lee, C.H. and Chung, J.H. (2001) J. Biol. Chem. 276, 30537-30541.

注意事项：

Storage: Chk2 siRNA I is supplied in RNAse-free water. Aliquot
and store at -20ºC.