Cell Signaling Technology

Product Pathways - Protein Stability

SignalSilence® Skp1 siRNA I #12514

No. Size Price
12514S 300 µl ( 3 nmol ) ¥3,224.00 现货查询 购买询价
12514 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
TFN Human,

Species cross-reactivity is determined by western blot.

Applications Key: TFN=Transfection,

Homology

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

Specificity / Sensitivity

SignalSilence® Skp1 siRNA I inhibits human, mouse, rat, and monkey Skp1 expression.

SignalSilence® Skp1 siRNA I抑制人、小鼠、大鼠和猴种Skp1的表达。

Description

SignalSilence® Skp1 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Skp1 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

来自Cell Signaling Technology (CST)的SignalSilence® Skp1 siRNA I可以帮助研究者通过RNA干扰特异性地抑制Skp1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。

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.

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。

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Skp1 siRNA I (+), using Skp1 (D3J4N) Rabbit mAb #12248 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The Skp1 (D3J4N) Rabbit mAb confirms silencing of Skp1 expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.Western blot检测转染有100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-)或SignalSilence® Skp1 siRNA I (+),采用Skp1 (D3J4N) Rabbit mAb #12248 (upper)或GAPDH (D16H11) XP® Rabbit mAb #5174 (lower)。Skp1 (D3J4N) Rabbit mAb证实了Skp1沉默表达,采用GAPDH (D16H11) XP® Rabbit mAb作为内参照。

Directions for Use

CST recommends transfection with 100 nM SignalSilence® Skp1 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.

CST推荐使用100 nM SignalSilence® Skp1 siRNA I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。

Background

Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2 and then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (1-3). Combinatorial interactions of different E2 and E3 proteins result in substrate specificity (4). Recent data suggests that activated E2 associates transiently with E3, and the dissociation is a critical step for ubiquitination (5). S phase kinase-associated protein 1 (Skp1) is a critical scaffold protein of the Skp1/CUL1/F-box (SCF) E3 ubiquitin ligase protein complex. Various F-box proteins (e.g., β-TrCP, Skp2) mediate an interaction with Skp1, via their defining and conserved domain of 40 amino acids, and with substrates to be ubiquitinated (e.g., β-catenin, p27) (4).

泛素通过泛素化作用可以与许多细胞蛋白共价连接,这些蛋白可以被26S蛋白酶体降解。3种组分包含在蛋白-泛素共价连接过程。泛素首先通过形成具有活化组分E1的硫酯复合物被激活;活化的泛素随后转运到泛素载体蛋白E2,随后从E2到泛素连接酶E3进行最后的转运到epsilon-NH2的靶蛋白赖氨酸残基(1-3)。不同E2和E3蛋白的相互作用导致底物的特异性(4)。最近的数据表明活化的E2短暂地与E3相关,解离是去泛素化的重要步骤(5)。S期激酶相关蛋白(Skp1)是Skp1/CUL1/F-box (SCF) E3泛素连接酶蛋白复合物的一种重要的支架蛋白。多种F-box蛋白(例如:β-TrCP, Skp2)介导与Skp1的相互作用,经它们确定的40氨基酸的保守结构域,与被泛素化的底物相互作用(例如:β-catenin, p27) (4)。

  1. Ciechanover, A. (1998) EMBO J. 17, 7151-60.
  2. Hochstrasser, M. (2000) Nat. Cell Biol. 2, E153-7.
  3. Hochstrasser, M. (2000) Science 289, 563-4.
  4. DeSalle, L.M. and Pagano, M. (2001) FEBS Lett. 490, 179-89.
  5. Deffenbaugh, A.E. et al. (2003) Cell 114, 611-22.

Application References

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Protocols

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For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

SignalSilence is a registered trademark of Cell Signaling Technology, Inc.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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