SignalSilence? UCHL1 siRNA II

• 产品编号：CST-12415S      品牌：CST       原厂货号：12415S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

来自Cell Signaling Technology (CST)的SignalSilence^® UCHL1 siRNA II可以帮助研究者通过RNA干扰特异性地抑制UCHL1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推荐使用100 nM SignalSilence^® UCHL1 siRNA II进行转染，48到72小时后对细胞进行裂解。转染步骤按照转染试剂说明书提供的步骤进行。遇到任何使用方面的问题，请随时联系CST。每小瓶可供100次转染，每次转染量相当于在转染24孔板时，每孔总体积为300μl培养基中siRNA的终浓度为100nM。蛋白泛素化和去泛素化作用是由泛素酶(UBEs)和去泛素酶(DUBs)催化的可逆过程(1,2)。DUBs分为5个亚家族：USP, UCH, OTU, MJD和JAMM 。UCHL1, UCHL3, UCHL5/UCH37和BRCA-1-相关蛋白-1(BAP1)属于DUBs的泛素碳末端的水解酶(UCH)家族，它们均具有含有约230个氨基酸的保守的催化结构域(UCH结构域)。UCHL5和BAP1有独特的延长的碳末端尾。UCHL1在神经组织和睾丸中大量表达，而UCHL3的表达则广泛分布(3,4)。尽管UCHL1和UCHL3属于亲近的UCH家族，并具有大约53%的同源性，但它们的生化特性却不同，UCHL1结合单泛素，UCHL3则显示则对泛素和泛素样分子NEDD8显示双重特性。UCHL1 (PGP 9.5/PARK5)作为去泛素化酶和单泛素稳定剂。体外研究表明，UCHL1能够水解Ub羧基末端甘氨酸和靶蛋白上的赖氨酸ε-氨基团之间的异肽键。UCHL1也在泛素前和核糖体蛋白翻译为泛素融合物的翻译过程中起作用(5-7)。UCHL1缺陷的小鼠呈现痉挛状态，表明UCHL1的活性对于正常结构和神经肌肉连接的功能是必须的(5-7)。研究表明在许多人恶性肿瘤中缺乏UCHL1的表达，诸如前列腺、结直肠、肾和乳腺癌。研究者已经证实乳腺癌，缺乏UCHL1的表达，有助于UCHL1启动子的超甲基化(8)。尽管人恶性肿瘤中缺乏UCHL1的表达，在神经变性疾病如Parkinson's 和Alzheimer's中均可能存在UCHL1的突变(6,7)。

1. Nijman, S.M. et al. (2005) Cell 123, 773-86.
2. Nalepa, G. et al. (2006) Nat Rev Drug Discov 5, 596-613.
3. Leroy, E. et al. (1998) Nature 395, 451-2.
4. Kurihara, L.J. et al. (2001) Hum Mol Genet 10, 1963-70.
5. Todi, S.V. and Paulson, H.L. (2011) Trends Neurosci 34, 370-82.
6. Setsuie, R. and Wada, K. Neurochem Int 51, 105-11.
7. Day, I.N. and Thompson, R.J. (2010) Prog Neurobiol 90, 327-62.
8. Xiang, T. et al. (2012) PLoS One 7, e29783.

原厂资料：

Homology

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

Specificity / Sensitivity

SignalSilence^® UCHL1 siRNA II inhibits human and monkey UCHL1 expression.

Description

from Cell Signaling Technology (CST) allows the researcher to specifically inhibit UCHL1 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® UCHL1 siRNA II 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

Protein ubiquitination and deubiquitination are reversible processes catalyzed by ubiquitinating enzymes (UBEs) and deubiquitinating enzymes (DUBs) (1,2). DUBs are categorized into 5 subfamilies: USP, UCH, OTU, MJD, and JAMM. UCHL1, UCHL3, UCHL5/UCH37, and BRCA-1-associated protein-1 (BAP1) belong to the Ubiquitin C-terminal Hydrolase (UCH) family of DUBs, which all possess a conserved catalytic domain (UCH domain) of about 230 amino acids. UCHL5 and BAP1 have unique extended C-terminal tails. UCHL1 is abundantly expressed in neuronal tissues and testes, while UCHL3 expression is more widely distributed (3,4). Although UCHL1 and UCHL3 are the most closely related UCH family members with about 53% identity, their biochemical properties differ in that UCHL1 binds monoubiquitin and UCHL3 shows dual specificity toward both ubiquitin (Ub) and NEDD8, a Ub-like molecule. 
 
 UCHL1 (PGP 9.5/PARK5) functions as a deubiquitinating enzyme and monoubiquitin stabilizer. In vitro studies have demonstrated that UCHL1 can hydrolyze isopeptide bonds between the C-terminal glycine of Ub and the ε-amino group of lysine on target proteins. UCHL1 is also involved in the cotranslational processing of pro-ubiquitin and ribosomal proteins translated as ubiquitin fusions (5-7). Mice deficient in UCHL1 experience spasticity, suggesting that UCHL1 activity is required for the normal structure and function of the neuromuscular junction (5-7). Research studies have implicated loss of UCHL1 expression in numerous human malignancies, such as prostate, colorectal, renal, and breast carcinomas. Investigators have shown that in breast carcinomas, loss of UCHL1 expression can be attributed to hyper-methylation of the UCHL1 promoter (8). While loss of UCHL1 expression is implicated in human carcinogenesis, mutation of UCHL1 has been implicated in neurodegenerative diseases such as Parkinson's and Alzheimer's (6,7).

1. Nijman, S.M. et al. (2005) Cell 123, 773-86.
2. Nalepa, G. et al. (2006) Nat Rev Drug Discov 5, 596-613.
3. Leroy, E. et al. (1998) Nature 395, 451-2.
4. Kurihara, L.J. et al. (2001) Hum Mol Genet 10, 1963-70.
5. Todi, S.V. and Paulson, H.L. (2011) Trends Neurosci 34, 370-82.
6. Setsuie, R. and Wada, K. Neurochem Int 51, 105-11.
7. Day, I.N. and Thompson, R.J. (2010) Prog Neurobiol 90, 327-62.
8. Xiang, T. et al. (2012) PLoS One 7, e29783.

注意事项：

Storage: UCHL1 siRNA II is supplied in RNAse-free water.
Aliquot and store at -20ºC.