Cell Signaling Technology

Product Pathways - Screening Technologies

PTMScan® Phospho-T*PP Motif (T*PP) XP® Rabbit mAb Kit #5567

motif   phospho-threonine   threonine  

REACTIVITY

No. Size Price
5567S 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® T*PP Motif (T*PP) 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® (T*PP) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the T*PP motif (T* = phospho-threonine). More specifically, cells are lysed in a urea-containing buffer, cellular proteins are digested by proteases, and resulting peptides fractionated by reversed-phase solid-phase extraction. Peptides are then subjected to immunoaffinity purification using T*PP Motif (T*PP) XP® Rabbit mAb against the MAPK/CDK substrate motif T*PP coupled to protein A agarose beads. Unbound peptides are removed through washing, and phosphomotif-containing peptides are eluted with dilute acid. Reversed-phase extraction is performed on microtips to separate phosphopeptides from antibody and to concentrate them for LC tandem mass spectrometry. A Limited Use License allowing the use of the patented PTMScan method is provided with the kit. Note: The XP® monoclonal antibody in this kit was generated using our proprietary XMT® monoclonal technology.

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®(ST*P) (T*PP) Motif Antibody珠子可以用来特异性富集含T*PP模体的磷酸化肽段(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。这些肽段然后被抗MAPK/CDK底物模体T*PP的T*PP Motif (T*PP) XP® 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。注意:此试剂盒中的XP®单克隆抗体利用我们所有的XMT®单克隆技术开发。

Chart

Chart

Chart showing the proportions of underlying sequence motifs found in a PTMScan® study using T*PP motif antibody and OrbiTrap MS analysis. The primary motif is highlighted in white. Analysis of peptides from HeLa cells, untreated and nocodazole-treated, gave 236 non-redundant sites. 88% of these sites fit motif T*PP; 8% sites are from motif T*P, and 4% of the sites are from motif S*P.

图片显示使用T*PP模体抗体和Orbitrap MS分析进行的PTMScan研究中发现的序列模体的属性。主要模体用白色加亮显示。对未处理或nocodazole处理的HeLa细胞肽段的分析给出236个非冗余位点。这些位点中88%为T*PP,8%为T*P模体,4%为S*P模体。

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。

Western Blotting

Western Blotting

T*PP motif western blot analysis of extracts from HeLa cells, untreated or Nocodazole treated, using T*PP Motif (T*PP) XP® Rabbit mAb.

使用T*PP Motif (T*PP) XP® Rabbit mAb对来自未处理或Nocodazole处理的Hela细胞抽提液进行T*PP模体的Western Blot分析。

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 the protocol in the kit package for further details.

推荐使用PTMScan® IAP Buffer Plus Detergent,它可以减少非特异性反应。如果去垢剂干扰IAP®上清液的分析,可使用PTMScan® IAP缓冲液。更多信息请参看工具和包装中的方案手册。

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). A T*PP phosphorylation motif has been extracted from a large-scale proteomic data set on HeLa nuclei (2). Western blot and proteomic analysis shows that phosphorylation on this motif is sensitive to cell cycle control. Similar motifs are phosphorylated by MAPK and CDK families of serine/threonine protein kinases, which play important roles in proliferation and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (motifs PX(S/T)P and (S/T)PXK/R) (3-5). The T*PP Motif (T*PP) XP® Rabbit mAb contained in this kit, which binds to the motif T*PP, was developed by CST for use in the study and discovery of new kinase substrates

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)。T*PP磷酸化模体在HeLa细胞核的大规模蛋白组学中被提取(2)。Western blot和蛋白组学分析显示这个模体的磷酸化对细胞周期控制很敏感。类似模体被MAPK和CDK家族丝氨酸/苏氨酸蛋白激酶磷酸化,在增殖和细胞周期控制中发挥重要作用。这些激酶磷酸化有脯氨酸跟随的苏氨酸或丝氨酸(模体PX(S/T)P和(S/T)PXK/R)(3-5)。包含在试剂盒中的T*PP模体(T*PP) XP® Rabbit mAb可以结合T*PP,由CST公司开发,用以研究新的激酶底物。

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.

XP is a registered 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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