Cell Signaling Technology

Product Pathways - Screening Technologies

PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Kit #12810

caspase   motif  

REACTIVITY

No. Size Price
12810S 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® Cleaved Caspase Substrate Motif [DE(T/S/A)D] 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® 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® services, please visit www.cellsignal.com/services/index.html.

PTMScan®技术采用CST专有的基于抗体的肽段富集方法,利用一种特殊的bead偶联的抗体进行免疫沉淀并结合液相色谱(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。这些蛋白质翻译后修饰包括磷酸化(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、鉴定以及定量分析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 PTMScan® LC-MS/MS experiment using 1044 nonredundant tryptic peptides with carboxy-terminal aspartates derived from human HeLa cells treated with Staurosporine #9953 1 μM for 3 hours to induce apoptosis. Peptides were immunoprecipitated with PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Immunoaffinity Beads. The logo represents the relative frequency of amino acids in each position leading up to the carboxy-terminal aspartate.

Motif Logo出自PhosphoScan® LC-MS/MS实验,实验中使用1044个低丰度胰蛋白酶消化的带有羧基末端天冬氨酸的多肽,这些多肽来源于经1 μM Staurosporine #9953处理3小时后引发凋亡的人HeLa细胞。多肽是用PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Immunoaffinity Beads免疫沉淀得到。该Logo代表了在羧基末端天冬氨酸之前的每个位置上的氨基酸的相对频率。

Chart

Chart

This chart shows the underlying motif distribution within 1044 nonredundant tryptic peptides with a carboxy-terminal aspartate derived from an LC-MS/MS experiment using HeLa cells treated with Staurosporine #9953 1μM, (3 hr; +) and immunoprecipitated with PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Immunoaffinity Beads. Within the same data set, the proportion of peptides with D in the -3 position is 36%, and the proportion of peptides with E in the -2 position is 53%.

图片显示了PhosphoScan® LC-MS/MS实验中得到的motif分布,实验中使用1044个低丰度胰蛋白酶消化的带有羧基末端天冬氨酸的多肽,这些多肽来源于经1 μM Staurosporine #9953处理3小时后引发凋亡的人HeLa细胞。免疫沉淀中使用的是PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Immunoaffinity Beads。在相同的数据组中,-3位置是D的多肽比例是36%,-2位置是E的多肽比例是53%。

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,蛋白酶消化胞内蛋白,产生的多肽通过反相固相萃取纯化。这些肽段然后使用偶联到蛋白A琼脂糖珠的PTMScan® Motif Antibody进行免疫亲和纯化。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

Apoptosis is a physiological process resulting in a highly regulated, programmed form of cell death that is a normal part of growth and development in multicellular organisms. Aspartic acid-directed cysteine proteases, caspases, are central to the apoptotic mechanism (1). An intrinsic pathway initiates the apoptotic cascade from signals originating within the cell, such as DNA damage, and an extrinsic pathway initiates apoptosis in response to extracellular signals, like FasL. In either case, initiator caspases, such as caspase-8 and 9, begin cleaving downstream substrates that include the effector caspases, such as caspase-3, the most prominent executioner caspase (2,3). These effector caspases amplify the apoptotic cascade to target many critical proteins needed for normal cell function. Apart from its role in developmental biology, the regulation of apoptosis has broad implications for the study of cancer, autoimmune, and infectious diseases among others (4). In the human proteome, there are thousands of known or putative caspase cleavage sites and all are cleaved at an aspartic acid residue, or, in rare cases, a glutamic acid residue. The resultant fragments containing a carboxy-terminal aspartate generally have the XEXD motif with some variation (5). The PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Kit provides a unique and sensitive method for quantifying hundreds of cleaved caspase substrates, including caspase-3 and 7, NEDD1, HDAC3, MCM4, Notch1, eIF2B, raptor, Bid, Smad6, NFAT90, and PTEN among others.

凋亡是一个受到高度调控、细胞程序化死亡的生理过程,是多种细胞组织生长和发育的一种正常组成部分。Caspase 是天冬胺酸介导的半胱氨酸蛋白酶,是凋亡机制的中枢(1)。这个内在的通路能够从来源于细胞内的信号如DNA损伤启动凋亡级联反应。而外在的通路能够对细胞外的信号如FasL响应,启动凋亡。在内在和外在通路中,起始因子caspase包括多种效应分子caspase以及细胞死亡的初级执行者 caspase-3会酶切下游底物(2,3)。效应器caspase会放大凋亡级联反应调控许多对正常细胞功能至关重要的靶蛋白。除了在发育生物学中的作用,凋亡调控对于研究癌症、自身免疫疾病以及传染性疾病具有广泛影响(4)。成千上万的已知或推测的caspase酶切位点呈现在人类蛋白质组中,几乎都含有天冬氨酸残基位点的酶切,同样也有在Glu残基位点的酶切,但很少。生成的含有羧基末端天冬氨酸的片段通常含有XEXD motif,并伴随着一些变化(5)。PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Kit提供了一种独有的灵敏方法,以定量分析被caspase酶切的成百上千种底物,包括caspase-3 和 7, NEDD1, HDAC3, MCM4, Notch1, eIF2B, raptor, Bid, Smad6, NFAT90, 以及 PTEN 等。

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products


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

U.S. Patent No. 5,675,063.

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.

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

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