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Ubn-ubiquitinylated substrate

BML-UW0610-0025 25 µg 278.00 USD
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Product Details

Source:Protein components produced in E. coli. Synthesized enzymatically in vitro. Ubn-ubiquitinylated substrate protein was produced using wild type ubiquitin.
UniProt ID:P0CG47 (UBB), P0CG48 (UBC), P62979 (RPS27A), P62987 (UBA52)
Formulation:Liquid. In 50mM TRIS, pH 7.5, containing 150mM NaCl and 1mM DTT.
Purity:≥95% (SDS-PAGE)
Purity Detail:High molecular weight conjugates were separated from free ubiquitin by size exclusion chromatography.
Quality Control:SDS-PAGE: Multiple high molecular weight bands are observed for Ub conjugate reactions (Fig. 1).
Immunoblotting: Detection of high molecular weight bands with the polyubiquitin-chain reactive monoclonal antibody FK2 (Prod. No. BML-PW8810) demonstrates their polyubiquitinylated, rather than mono- or multiubiquitinylated, status (Fig. 2).
DUB reactivity: The potential utility of Ubn-ubiquitinylated conjugates as DUB substrates was demonstrated by their deconjugation in the presence of the isolated catalytic domain of USP2 (Prod. No. BML-UW9850), a DUB of general and promiscuous activity that appears to exhibit no linkage preference (Fig. 3).
Application Notes:Uses:
1. Deubiquitinylating enzyme substrates (general/linkage specific).
2. Profiling of DUB linkage-type preference or specificity in combination with other single lysine only polyubiquitinylated substrates (e.g. (K11-only)Ubn-ubiquitinylated substrate, Prod. No. BML-UW0620)
3. Investigation of polyubiquitin chain recognition by and interaction with ubiquitin binding proteins.
Shipping:Shipped on Dry Ice
Short Term Storage:+4°C
Long Term Storage:-80°C
Use/Stability:Stable for at least 6 months after receipt when stored at -80°C.
Handling:Avoid freeze/thaw cycles. After opening, prepare aliquots and store at -80°C.
Scientific Background:Polyubiquitin chains, linked through specific lysine residues, are useful tools for investigating the specificity and reactivity of deubiquitinylating enzymes (DUBs) and the recognition and interaction of polyubiquitin modified proteins with ubiquitin binding domain (UBDs) containing proteins.
Technical Info/Product Notes:TECHNICAL NOTE: Under certain conditions these polyubiquitinylated substrates can bind to metal affinity matrices. This may have implications for their successful use in some applications, for example, if they are to be used in concert with HIS-tagged proteins requiring subsequent affinity isolation with metal ion affinity resins. Such methods of detection or isolation should be avoided wherever possible in order to avoid complication in interpretation of results obtained.
Regulatory Status:RUO - Research Use Only
Ubn-ubiquitinylated substrate SDS-PAGE
Fig 1:SDS-PAGE analysis. Lane 1: MW Marker, Lane 2: 2 μg, Lane 3: 5 μg, Lane 4: 10 μg BML-UW0610
Ubn-ubiquitinylated substrate Western blot
Fig 2:Western Blot analysis. Lane 1: MW Marker, Lane 2: Substrate treated with USP2, Lane 3: Substrate without protease. Probed with Ubiquitin linkage-specific mAb (Prod. No. BML-PW8810).
Ubn-ubiquitinylated substrate DUB_assay
Fig 3:DUB assay. Substrate (Prod. No. BML-UW0610) was incubated with USP2 (Prod. No. BML-UW9850). Lane 1: MW Marker, Lane 2: Substrate without protease, Lane 3: Substrate with protease, Lane 4: Protease without substrate.
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Ubn-ubiquitinylated substrate SDS-PAGE Ubn-ubiquitinylated substrate Western blot Ubn-ubiquitinylated substrate DUB_assay

General Literature References

Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex: L. Jin, et al.; Cell 133, 653 (2008), Abstract;
Proteasome-independent functions of ubiquitin in endocytosis and signaling: D. Mukhopadhyay & H. Riezman; Science 315, 201 (2007), Abstract;
Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting: A. Ciechanover; Exp. Biol. Med. (Maywood) 231, 1197 (2006), Abstract;
Distinct monoubiquitin signals in receptor endocytosis: K. Haglund, et al.; Trends Biochem. Sci. 28, 598 (2003), Abstract;
The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin: F. Wu-Baer, et al.; J. Biol. Chem. 278, 34743 (2003), Abstract;
The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction: M.H. Glickman & A. Ciechanover; Physiol. Rev. 82, 373 (2002), Abstract;

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