Motif analysis was done using all ubiquitin remnant peptides in a ubiquitin remnant motif antibody PTMScan® study. Cellular tryptic peptides from HCT 116 cells, untreated or treated with MG-132 #2194, were immunoprecipitated with ubiquitin remnant motif antibody K-ε-GG and analyzed by OrbiTrap™ LC-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.
|Product Includes||Cap Color||Volume (with Count)|
|PTMScan® Ubiquitin Branch Motif (K-ε-GG) Immunoaffinity Beads||Blue||3 x 80 µl|
|PTMScan® IAP Buffer (10X) 9993||White||3 x 600 µl|
|PTMScan® Trypsin Digested Control Peptides I 12219||Blue||1 x 1 vial|
|PTMScan® Limited Use License||1 x license|
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 are included with the kit.
PTMScan® Trypsin Digested Control Peptides I are supplied as lyophilized powder. Centrifuge the tube containing the lyophilized peptide in order to collect all material to be dissolved. Add 1.4 ml PTMScan® IAP Buffer (1X) and follow the PTMScan® kit protocol from the Immunoaffinity Purification (IAP) step using the entire vial of reconstituted peptides.
Antibody beads are supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody. Store lyophilized peptides at -20°C. In lyophilized form, the peptides are stable for 24 months.
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.
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 the ubiquitin-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 ubiquitin 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 analysis for quantitative profiles of hundreds to over a thousand non-redundant ubiquitinated sequences.
AcetylScan is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
MethylScan is a trademark of Cell Signaling Technology, Inc.
PhosphoScan 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.
OrbiTrap is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
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 firstname.lastname@example.org.