Product Pathways - Screening Technologies
PTMScan® Phospho-AMPK Substrate Motif (LXRXXS*/T*) Kit #5564
|5564S||1 Kit ( 5 assays )||email@example.com|
|Products Included||Quantity||Cap Color|
|PTMScan® (LXRXXS*/T*) Motif Antibody Bead Conjugate||5 x 80 µl|
|PTMScan® IAP Buffer (10X)||5 x 0.6 ml||White|
|PTMScan® Limited Use License|
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® 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 is included with the kit.
PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology 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 (1). For more information on PTMScan® services, please visit www.cellsignal.com/services/index.html.
AMPK substrate motif western blot analysis of extracts from H1650 cells, untreated or Phenformin treated using AMPK Substrate Motif (LXRXXS */T*) Rabbit mAb.
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.
The Motif Logo shows the amino acid distributions around the sites recognized by the antibody.
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.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).
- Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.
- Hardie, D.G. (2004) J Cell Sci 117, 5479-87.
- Carling, D. (2004) Trends Biochem Sci 29, 18-24.
- Gwinn, D.M. et al. (2008) Mol Cell 30, 214-26.
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For Research Use Only. Not For Use In Diagnostic Procedures.