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92465
PTMScan® Sumoylation Remnant Motif Kit
Proteomic Analysis Products
PTMScan

PTMScan® Sumoylation Remnant Motif Kit #92465

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PTMScan® Sumoylation Remnant Motif Kit: Image 1

Motif analysis was done using all SUMO remnant peptides in a SUMO remnant motif antibody PTMScan® study. Cellular WaLP peptides from HeLa cells or treated with heat shock, were immunoprecipitated with SUMO remnant motif antibody K-ε-GG and analyzed by OrbiTrap MS. The study gave 841 non-redundant sites. The Motif logo reflects the relative prevalence of an amino acid in each position relative to the background in the human proteome. Residues represented above the x-axis are enriched relative to their expected frequency in this background. For more information on motif analysis using PSP, please visit www.phosphosite.org.

Product Includes Volume (with Count)
PTMScan® Branch Motif (K-ε-GG) Immunoaffinity Beads 10 x 80 µl
PTMScan® IAP Buffer (10X) 9993 10 x 600 µl
PTMScan® Wild Type Alpha-Lytic Protease (WaLP) 33036 4 x 400 µg
PTMScan® Limited Use License 1 x 1 ml

Product Usage Information

Important: Wild type alpha-lytic protease (WaLP) is a serine endopeptidase that cleaves at the carboxyl terminal side of amino acids alanine, serine, threonine, and valine. Please check that the predicted SUMO sequence of your model organism contains AGG, SGG, TGG or VGG at the c-terminus to ensure reactivity.

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. A detailed protocol and Limited Use License allowing the use of the patented PTMScan® method is included with the kit.

Storage

Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.

Product 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® Technology 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® Proteomics Services, please visit www.cellsignal.com/services/index.html

Background

Small ubiquitin-related modifier 1, 2, and 3 (SUMO-1, -2, and -3) are members of the ubiquitin-like protein family (1). The covalent attachment of the SUMO-1, -2, or -3 (SUMOylation) to target proteins is analogous to ubiquitination. This post-translational modification is a reversible, multi-step process that is initiated by cleaving a precursor protein to a mature protein. Mature SUMO-1, -2, or -3 is then linked to the activating enzyme E1 and conjugated to E2. In conjunction with E3, SUMO-1, -2, or -3 is ligated to the target protein (2). Ubiquitin and the individual SUMO family members are all targeted to different proteins with diverse biological functions. Ubiquitin predominantly regulates degradation of its target (1). In contrast, SUMO-1 is conjugated to RanGAP, PML, p53, and IκB-α to regulate nuclear trafficking, formation of subnuclear structures, regulation of transcriptional activity, and protein stability (3-7). SUMO-2/-3 forms poly-(SUMO) chains, is conjugated to topoisomerase II and APP, regulates chromosomal segregation and cellular responses to environmental stress, and plays a role in the progression of Alzheimer disease (8-11).

In this assay, PTMScan® Branch Motif (K-ε-GG) Immunoaffinity Beads, a proprietary branch (“K-ε-GG”) antibody with specificity for a di-glycine tag that is the remnant of SUMO left on protein substrates after WaLP digestion, is used to enrich sumoylated peptides from WaLP-digested cell samples. This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand non-redundant sumoylated sequences.

  1. Schwartz, D.C. and Hochstrasser, M. (2003) Trends Biochem Sci 28, 321-8.
  2. Kim, K.I. et al. (2002) J Cell Physiol 191, 257-68.
  3. Matunis, M.J. et al. (1996) J Cell Biol 135, 1457-70.
  4. Duprez, E. et al. (1999) J Cell Sci 112 ( Pt 3), 381-93.
  5. Gostissa, M. et al. (1999) EMBO J 18, 6462-71.
  6. Rodriguez, M.S. et al. (1999) EMBO J 18, 6455-61.
  7. Desterro, J.M. et al. (1998) Mol Cell 2, 233-9.
  8. Tatham, M.H. et al. (2001) J Biol Chem 276, 35368-74.
  9. Azuma, Y. et al. (2003) J Cell Biol 163, 477-87.
  10. Li, Y. et al. (2003) Proc Natl Acad Sci U S A 100, 259-64.
  11. Saitoh, H. and Hinchey, J. (2000) J Biol Chem 275, 6252-8.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
AcetylScan is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
CST is a trademark of Cell Signaling Technology, Inc.
MethylScan is a trademark of Cell Signaling Technology, Inc.
PhosphoSitePlus is a trademark of Cell Signaling Technology, Inc.
PTMScan is a trademark of Cell Signaling Technology, Inc.
UbiScan is a trademark of Cell Signaling Technology, Inc.
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 [email protected] For information regarding commercial licensing terms please contact CST Pharma Services Department at [email protected]