Product Pathways - Protein Stability
PTMScan® Ubiquitin Remnant Motif (K-ε-GG) Kit #5562
|5562S||1 Kit ( 5 assays )||firstname.lastname@example.org|
|Products Included||Quantity||Cap Color|
|PTMScan® Ubiquitin Remnant Motif (K-ε-GG) 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.
Motif analysis was done using all ubiquitin remnant peptides in a ubiquitin remnant motif antibody PTMScan® study. Cellular tryptic peptides from HCT116 cells. untreated or treated with MG132, were immunoprecipitated with ubiquitin remnant motif antibody K-ε-GG and analyzed by OrbiTrap MS. The study gave 599 non-redundant sites. The motif logo shows that the K-ε-GG antibody is a general motif antibody that recognizes the K-ε-GG motif and does not have other amino acid preferences.
Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to theubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (2-4). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25a and bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (5-8).In this assay, the PTMScan® Ubiquitin Remnant Motif (K-ε-GG) Antibody Bead Conjugate, a proprietary ubiquitin branch (“K-ε-GG”) antibody with specificity for a di-glycine tag that is the remnant of ubiqui- tin left on protein substrates after trypsin digestion, is used to enrich ubiquitinated peptides from trypsin digested cell samples. This enrichment is followed by LC-MS/MS analy- sis for quantitative profiles of hundreds to over a thousand non-redundant ubiquitinated sequences.
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- Salomoni, P. and Pandolfi, P.P. (2002) Nat Cell Biol 4, E152-3.
- Jesenberger, V. and Jentsch, S. (2002) Nat Rev Mol Cell Biol 3, 112-21.
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For Research Use Only. Not For Use In Diagnostic Procedures.