Cell Signaling Technology

Product Pathways - Screening Technologies

PTMScan® Phospho-Akt Substrate Motif mAb 1 (RXXS*/T*) Kit #5561

akt1   akt2   akt3   motif  

REACTIVITY

No. Size Price
5561S 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® Phospho-Akt Substrate Motif (RXXS*/T*) Immunoaffinity Beads 80 µl Rabbit
PTMScan® IAP Buffer (10X) #9993 600 µl
PTMScan® Limited Use License license

Description

PTMScan® technology allows for the identification of post-translational modification sites in cellular proteins by utilizing immunoprecipitation in conjunction with liquid chromatography (LC) tandem mass spectrometry (MS). In this assay, PTMScan® (RXXS*/T*) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the RXXS*/T* motif (S*= phospho-serine, T*= phospho-threonine). More specifically, cells are lysed in a urea-containing buffer, cellular proteins are digested by proteases, and resulting peptides are fractionated by reversed-phase solid-phase extraction. Peptides are then subjected to immunoaffinity purification using Phospho-Akt Substrate (RXXS/T) Rabbit mAb 1 coupled to protein A agarose beads. Unbound peptides are removed through washing, and phospho-motif-containing peptides are eluted with dilute acid. Reversed-phase chromatography is performed on microtips to separate phosphopeptides from antibody and to concentrate them for LC tandem MS. A Limited Use License allowing the use of the patented PTMScan method is provided with the kit.

PTMScan®技术利用免疫共沉淀结合液相色谱串联质谱来识别细胞内蛋白质的转录后修饰位点。在这个测试中PTMScan® (RXXS*/T*) Motif Antibody珠子可以用来特异性富集含RXXS*/T*模体的磷酸化肽段(S*是磷酸化丝氨酸,T*是磷酸化苏氨酸)。更特异地,细胞在含尿素的缓冲液中裂解,细胞内蛋白被蛋白被消化,产生被反相固相萃取的肽段碎片。这些肽段然后被Phospho-Akt Substrate (RXXS/T) Rabbit mAb结合到蛋白A琼脂糖珠上免疫亲和纯化。未结合的抗体被清洗时去除,包含磷酸化模体的肽段被稀释的酸洗脱。微探尖上进行反相固相萃取分离磷酸化肽段和抗体,然后浓缩以便进行液相色谱串联质谱。一个有限使用许可批准了使用这个试剂盒中的受专利保护的PTMScan方法。

Chart

Chart

Chart showing the proportions of underlying Akt substrate-like sequence motifs found in an Akt substrate PTMScan® study using Akt Substrate Motif mAb 1. Analysis of three cell lines gave 281 non-redundant peptide sequences containing Akt substrate-like motifs (Moritz et al., Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tryosine kinases. Sci. Signal. 2010, 3, ra64). The proportion containing phosphoserine was 74%, and the proportion with arginine residues are both the -5 and -3 positions was 36%. Although this antibody has a preference for arginine residues at the -5 and -3 positions, it also recognizes peptides with an arginine residue at just the -3 position.

图片显示使用Akt Substrate Motif mAb 1进行的AKt底物PTMScan研究中发现的Akt底物样序列模体的属性. 对着三个细胞系的分析给出了281个包含Akt底物样模体的非冗余肽段序列(Moritz等,Akt-RSK-S6激酶信号通路网络被致癌基因受体酪氨酸激酶激活,Sci. Signal. 2010, 3, ra64)。包含磷酸化丝氨酸的比率为74%,在-5和-3位有精氨酸残基的比率为36%。尽管此抗体对-5和-3位有精氨酸的模体有偏好,它也能识别仅在-3位有精氨酸残基的肽段。

Motif Logo

Motif Logo

The Motif Logo shows the amino acid distributions around the sites recognized by the antibody.

模体标志图显示出分布在此抗体识别位点周围的氨基酸。

Directions for Use

The use of PTMScan® IAP Buffer Plus Detergent is recommended; it may reduce non-specific interactions. If detergent interferes with analyses to be performed on the IAP supernatant, use PTMScan® IAP Buffer. Please see protocol for further details.

推荐使用PTMScan® IAP Buffer Plus Detergent,它可以减少非特异性反应。如果洗涤剂干扰IAP®上清液的分析,可使用PTMScan® IAP缓冲液。更多信息请参看方案手册。

Background

PTMScan® technology employs a proprietary methodology from Cell Signaling Technology (CST) for antibody-based peptide enrichment combined with tandem mass spectrometry for quantitative profiling of post-translational modifications (PTMs), including phosphorylation (PhosphoScan®), ubiquitination (UbiScan®) and acetylation (AcetylScan®). PhosphoScan enables researchers to isolate a large number of cellular phosphopeptides, providing a global overview of phosphorylation in cell and tissue samples. This technology allows for the exploration of phosphorylation events, without preconceived biases about where phosphorylation sites will be found. PhosphoScan employs phospho-residue (Tyr, Ser, Thr) motif antibodies for phosphopeptide immunoaffinity purification from cell extracts combined with LC tandem MS to identify and quantify changes in phosphorylation levels (1). Akt plays a central role in mediating critical cellular responses including cell growth and survival, angiogenesis, and transcriptional regulation (2-4). It is a member of an important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, which includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, p70 S6 kinase, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (5,6). Akt, p70S6 kinase and RSK additionally share specificity for Arg at position -5 (recognition sequence RXRXXS/T) (7) In a recent phosphoproteomic study (8) co-authored by scientists in the CST Site Discovery Group over 300 downstream substrates for AGC family kinases recognizing the RXRXXS/T motif were identified with PhosphoScan Technology using Phospho-Akt substrate antibodies. These CST™ antibodies are powerful tools for investigating the regulation of phosphorylation by Akt and other Arg-directed kinases, as well as for high throughput kinase drug discovery.

PTMScan®技术采用CST公司独有的基于抗体的肽段富集方法,结合串联质谱定量分析翻译后修饰(PTM),包括磷酸化(PhosphoScan®)、泛素化(UbiScan®)和乙酰化(AcetylScan®)。PhosphoScan可以是研究者能够分离大量细胞内磷酸化肽,对细胞和组织的磷酸化情况有全局的认识。此技术使得我们可以探索磷酸化事件,不带有关于磷酸化位点在何处的先入为主的偏见。PhosphoScan采用磷酸化残基(Tyr、Ser、Thr)模体抗体对细胞抽提物进行磷酸化肽免疫亲和纯化,结合液相色谱串联质谱识别定量描述磷酸化水平的改变(1)。Akt在介导细胞内关键反应中扮演重要角色,包括细胞生长和生存、血管生成和转录调节(2-4)。精氨酸导向的激酶或AGC家族激酶是很重要的激酶,包括cAMP依赖的蛋白激酶(PKA)、cGMP依赖的蛋白激酶(PKG)、蛋白激酶C、Akt和RSK。这些激酶有相同的底物特异性,-3位上有精氨酸的丝氨酸和苏氨酸(5,6)。Akt,p70S6激酶和RSK共享对-5位精氨酸的特异性(识别RXRXXS/T序列)(7)。在最近的由CST Site Discovery Group研究组科学家进行的研究(8)中,利用磷酸化Akt底物抗体和PhosphoScan技术,对识别RXRXXS/T模体的AGC家族下游磷酸化蛋白进行了蛋白组学研究,识别出了超过300个下游底物。这些抗体由Cell Signaling Technology公司建立,是研究Akt磷酸化调节和其它精氨酸导向的激酶的有力工具,也可以用于高通量药物发现。

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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