Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

PTMScan® Mono-Methyl Arginine Motif [mme-RG] Kit #12235

REACTIVITY

No. Size Price
12235S 1 Kit ( 10 assays ) 请询价 现货查询 购买询价
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
PTMScan® Mono-Methyl Arginine Motif [mme-RG] Immunoaffinity Beads 80 µl Rabbit IgG
PTMScan® IAP Buffer (10X) #9993 600 µl
PTMScan® Limited Use License license

Description

PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology (CST) for peptide enrichment by immunoprecipitation using a specific bead-conjugated antibody in conjunction with liquid chromatography (LC) tandem mass spectrometry (MS/MS) for quantitative profiling of post-translational modification (PTM) sites in cellular proteins. These include phosphorylation (PhosphoScan®), ubiquitination (UbiScan®), acetylation (AcetylScan®), and methylation (MethylScan®), among others. PTMScan® Technology enables researchers to isolate, identify, and quantitate large numbers of post-translationally modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of PTMs in cell and tissue samples without preconceived biases about where these modified sites occur. For more information on PTMScan® Proteomics Services, please visit www.cellsignal.com/services/index.html.PTMScan®技术使用的是CST用于多肽富集的专利技术,利用免疫共沉淀通过一种特殊的与bead偶联的抗体结合液相色谱(LC)串联质谱(MS/MS)来定量分析细胞内蛋白质翻译后修饰(PTM)位点。此外还有磷酸化(PhosphoScan®)、泛素化(UbiScan®)、乙酰化(AcetylScan®)以及甲基化 (MethylScan®)。 PTMScan®技术可以使研究者能够分离、鉴定以及定量许多翻译后修饰的细胞内多肽,并具有很高的特异性和灵敏度,从而了解细胞和组织样品中PTM的全面概况,并且不带有任何关于这些修饰位点位于何处的先入为主的偏见。更多有关PTMScan® Proteomics Services的信息,请访问www.cellsignal.com/services/index.html.

Motif Logo

Motif Logo

The Motif Logo was generated from a MethylScan® LC-MS/MS experiment using 722 nonredundant tryptic peptides derived from human HCT 116 cells immunoprecipitated with PTMScan® Mono-Methyl Arginine motif [mme-RG] Immunoaffinity Beads. The logo represents the relative frequency of amino acids in each position surrounding the central methylated arginine residue. Glycine residues are enriched, especially at the +1 position, in the context of mono-methylated arginine when compared to the overall expected frequency in the human proteome. Of the total methylated arginine peptides, 68% contain the [mme-RG] motif.

Motif Logo出自MethylScan® LC-MS/MS实验,实验中使用722种低丰度胰蛋白酶消化的多肽,这些来源于人HCT 116细胞的多肽,是使用PTMScan® Mono-Methyl Arginine motif [mme-RG] Immunoaffinity Beads免疫沉淀得到的。该Logo代表了围绕中心的甲基化精氨酸残基的每个位置上氨基酸的相对频率。当与人蛋白质组整个预期的频率比较时,在单甲基化精氨酸的背景下甘氨酸残基被富集,尤其是+1位置。在全部精氨酸甲基化的多肽中,68%含有[mme-RG] 基序。

Chart

Chart

The pie chart shows the relative category distribution of proteins with mono-methylated arginine identified from peptides generated from a MethylScan® LC-MS/MS experiment of HCT 116 cells using PTMScan® Mono-Methyl Arginine (mme-RG) Immunoaffinity Beads.

饼图显示了从鉴定的多肽中含有单甲基化精氨酸的蛋白的相对种类分布,多肽来自MethylScan® LC-MS/MS实验,使用PTMScan® Mono-Methyl Arginine (mme-RG) Immunoaffinity Beads从HCT116 细胞中免疫得到。

Directions for Use

Cells are lysed in a urea-containing buffer, cellular proteins are digested by proteases, and the resulting peptides are purified by reversed-phase solid-phase extraction. Peptides are then subjected to immunoaffinity purification using a PTMScan® Motif Antibody conjugated to protein A agarose beads. Unbound peptides are removed through washing, and the captured PTM-containing peptides are eluted with dilute acid. Reversed-phase purification is performed on microtips to desalt and separate peptides from antibody prior to concentrating the enriched peptides for LC-MS/MS analysis. CST recommends the use of PTMScan® IAP Buffer #9993 included in the kit. An alternate PTMScan® IAP Buffer Plus Detergent #9992, which may reduce nonspecific interactions, is available separately. A detailed protocol and Limited Use License allowing the use of the patented PTMScan® method are included with the kit.使用含尿素的缓冲液裂解细胞,细胞内蛋白被蛋白酶消化,产生的多肽通过反相固相萃取纯化。然后使用偶联到蛋白A琼脂糖珠的PTMScan® Motif Antibody免疫亲和纯化这些肽段。未结合的多肽通过清洗去除,捕捉到的含有PTM的肽段使用稀释的酸进行洗脱。在浓缩富集到的多肽用于LC-MS/MS 分析前,需要在microtip上进行反相纯化以脱盐并将多肽从抗体中分离。CST推荐使用试剂盒中提供的PTMScan® IAP Buffer #9993。另外还可以使用PTMScan® IAP Buffer Plus Detergent #9992缓冲液,它可以减少非特异性反应,可以单独购买。有关PTMScan®专利方法的详细操作步骤以及使用许可限制可在试剂盒中查询。

Background

Arginine methylation is a prevalent PTM found on both nuclear and cytoplasmic proteins. Arginine methylated proteins are involved in many different cellular processes, including transcriptional regulation, signal transduction, RNA metabolism, and DNA damage repair (1-3). Arginine methylation is carried out by the arginine N-methyltransferase (PRMT) family of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a guanidine nitrogen of arginine (4). There are three different types of arginine methylation: asymmetric dimethylarginine (aDMA, omega-NG,NG-dimethylarginine), where two methyl groups are placed on one of the terminal nitrogen atoms of the guanidine group of arginine; symmetric dimethylarginine (sDMA, omega-NG,N’G-dimethylarginine), where one methyl group is placed on each of the two terminal guanidine nitrogens of arginine; and monomethylarginine (MMA, omega-NG-dimethylarginine), where a single methyl group is placed on one of the terminal nitrogen atoms of arginine. Each of these modifications has potentially different functional consequences. Though all PRMT proteins catalyze the formation of MMA, Type I PRMTs (PRMT1, 3, 4, and 6) add an additional methyl group to produce aDMA, while Type II PRMTs (PRMT5 and 7) produce sDMA. Methylated arginine residues often reside in glycine-arginine rich (GAR) protein domains, such as RGG, RG, and RXR repeats (5). However, PRMT4/CARM1 and PRMT5 methylate arginine residues within proline-glycine-methionine rich (PGM) motifs (6).精氨酸甲基化是常见的翻译后修饰,在核和细胞质蛋白中均有发现。精氨酸甲基化蛋白涉及许多细胞过程,包括转录后调节、信号转导、RNA代谢和DNA损伤修复(1-3)。精氨酸甲基化由精氨酸N甲基转移酶(PRMT)家族催化甲基从S-腺苷甲硫氨酸(AdoMet)转移到精氨酸胍氮上而形成(4)。精氨酸甲基化有三种形式:不对称的二甲基化(aDMA, omega-NG,NG-二甲基精氨酸),两个甲基基团在精氨酸胍基氮原子的一端;对称的二甲基化(sDMA, omega-NG, N'G-二甲基精氨酸),每个甲基基团在精氨酸胍基氮原子的一端;单甲基化(MMA, omega-NG-二甲基精氨酸),只有一个甲基集团在精氨酸胍基氮原子上。每种修饰都有其潜在的不同功能。尽管所有PRMT蛋白都催化MMA的形成,I型PRMT(PRMT1, 3, 4, 6)可以添加一个额外的甲基基团从而产生aDMA,II型PRMT(PRMT5和7)产生sDMA。甲基化精氨酸残基经常在富含甘氨酸-精氨酸(GAR)蛋白结构域中出现,如RGG, RG, RXR重复序列(5)。然而,PRMT4/CARM1和PRMT5可以甲基化含脯氨酸-甘氨酸-蛋氨酸(PGM)基序的精氨酸序列(6)。

  1. Bedford, M.T. and Richard, S. (2005) Mol Cell 18, 263-72.
  2. Pahlich, S. et al. (2006) Biochim Biophys Acta 1764, 1890-903.
  3. Bedford, M.T. and Clarke, S.G. (2009) Mol Cell 33, 1-13.
  4. McBride, A.E. and Silver, P.A. (2001) Cell 106, 5-8.
  5. Gary, J.D. and Clarke, S. (1998) Prog Nucleic Acid Res Mol Biol 61, 65-131.
  6. Cheng, D. et al. (2007) Mol Cell 25, 71-83.

Application References

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Companion Products


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

Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at ptmscan@cellsignal.com.

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

PTMScan is a trademark of Cell Signaling Technology, Inc.

AcetylScan is a trademark of Cell Signaling Technology, Inc.

UbiScan is a trademark of Cell Signaling Technology, Inc.

MethylScan is a trademark of Cell Signaling Technology, Inc.

U.S. Pat. No. 9,085,609

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

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