Cell Signaling Technology

Product Pathways - Screening Technologies

PTMScan® Phospho-AMPK Substrate Motif (LXRXXS*/T*) Kit #5564

AMPK   AMPK a   AMPK alpha   AMPKa   AMPKalpha   motif   pampk  

REACTIVITY

No. Size Price
5564S 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-AMPK Substrate Motif (LXRXXS*/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® (LXRXXS*/T*) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the (LXRXXS*/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 fractionated by reversed-phase solid-phase extraction. Peptides are then subjected to immunoaffinity purification using AMPK Substrate Motif (LXRXXS */T*) Rabbit mAb 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.

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。在这个检测中PTMScan® (LXRXXS*/T*) Motif Antibody基序抗体珠可以用来特异性富集含LXRXXS*/T*基序的磷酸化肽段(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,细胞内蛋白被蛋白酶消化,产生被反相固相萃取的肽段碎片。这些肽段然后被AMPK Substrate Motif (LXRXXS */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,包含磷酸化基序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。一个有限使用许可证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 proportion of underlying sequence motifs found in a PTMScan® study using AMPK substrate antibody and Orbitrap LC-MS analysis. Analysis of peptides from H1650 cells gave 187 non-redundant sites containing AMPK substrate and related motifs. The primary motifs are highlighted in white. 83% of these sites contain phosphoserine, and the proportion with leucine or methionine residues at the -5 position and arginine at the -3 position (AMPK substrate motif) was 41%. Although this antibody has a preference for the AMPK substrate motif, it also recognizes peptides with just an arginine residue at the -3 position.

图片显示使用AMPK底物抗体和Orbitrap LC-MS分析进行的PTMScan研究中发现的序列模体的属性. 对H1650细胞肽段的实验分析给出包含AMPK底物和相关模体的187个非冗余位点。主要模体用白色加亮显示。包含磷酸化丝氨酸的比率为83%,在-5位有亮氨酸和蛋氨酸和-3位有精氨酸残基(AMPK底物模体)的比率为41%。尽管此抗体对AMPK底物模体有偏好,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。

Western Blotting

Western Blotting

AMPK substrate motif western blot analysis of extracts from H1650 cells, untreated or Phenformin treated using AMPK Substrate Motif (LXRXXS */T*) Rabbit mAb.

使用AMPK Substrate Motif (LXRXXS */T*) Rabbit mAb对来自未处理或Phenformin处理的H1650细胞抽提液进行Akt底物模体的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). AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia, and ischemia (2). Several mutations in AMPKγ subunits have been identified, most of which are located in the putative AMP/ATP binding sites (CBS or Bateman domains). Mutations at these sites lead to reduction of AMPK activity and cause glycogen accumulation in heart or skeletal muscle (2,3). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (2). 
 
 AMPK Substrate Motif (LXRXXS */T*) Rabbit mAb contained in this kit, which binds to the AMPK phosphorylation motif LXRXXS*/T* (4), was developed by CST for use in the study and discovery of new AMPK 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)基序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)。1AMP激活的蛋白激酶(AMPK)从酵母到动植物高度保守,在调节能量平衡上发挥关键作用(2)。该激酶被细胞或环境应激时升高的AMP/ATP比率激活,如热休克、缺氧和缺血(2)。AMPKγ的若干突变已被确认,大部分都定位于推测的AMP/ATP结合位点(CBS或Bateman区域)。这些位点的突变导致AMPK活性的降低,导致心脏和骨骼肌中的糖原堆积(2,3)。越来越多的证据显示AMPK不仅调节脂肪酸和糖原代谢,也通过EF2和TSC2/mTOR通路调节蛋白质合成和细胞生长,通过eNOS/nNOS通路调节血流(2)。包含在试剂盒中的AMPK Substrate Motif (LXRXXS */T*) Rabbit mAb可以结合AMPk磷酸化基序LXRXXS*/T*(4),由CST公司开发,用以研究新的AMPK底物。

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.

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.

用户评论 --- 共 0

该产品暂无评论!

我要参与评论 :

如要参与评论请先登录网站

还没有网站账户?去注册一下吧

Products

 

Applications