Product Information
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
Specificity / Sensitivity
Species predicted to react based on 100% sequence homology
The antigen sequence used to produce this antibody shares
100% sequence homology with the species listed here, but
reactivity has not been tested or confirmed to work by CST.
Use of this product with these species is not covered under
our
Product Performance Guarantee.
All Species Expected
Product Description
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.
- Schwartz, D.C. and Hochstrasser, M. (2003) Trends Biochem Sci 28, 321-8.
- Kim, K.I. et al. (2002) J Cell Physiol 191, 257-68.
- Matunis, M.J. et al. (1996) J Cell Biol 135, 1457-70.
- Duprez, E. et al. (1999) J Cell Sci 112 ( Pt 3), 381-93.
- Gostissa, M. et al. (1999) EMBO J 18, 6462-71.
- Rodriguez, M.S. et al. (1999) EMBO J 18, 6455-61.
- Desterro, J.M. et al. (1998) Mol Cell 2, 233-9.
- Tatham, M.H. et al. (2001) J Biol Chem 276, 35368-74.
- Azuma, Y. et al. (2003) J Cell Biol 163, 477-87.
- Li, Y. et al. (2003) Proc Natl Acad Sci U S A 100, 259-64.
- Saitoh, H. and Hinchey, J. (2000) J Biol Chem 275, 6252-8.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Cross-Reactivity Key
H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected
Trademarks and Patents
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