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([K6-only]Ub)n-ubiquitinylated substrate

BML-UW0615-0025 25 µg 230.00 USD
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Product Specification

Source:Protein components produced in E. coli. Synthesized enzymatically in vitro. (K6-only)Ubn-ubiquitinylated substrate protein was produced using K6-only mutant 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 (K6-only) 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).
Mass spectrometry: Conjugates were analyzed by mass spectrometry following tryptic digest. A signature peptide corresponding to the predicted branched ubiquitin fragments (H-MQIFVK(G-G)TLTGK-OH) was identified, confirming the polyubiquitinylated status of the conjugates and the nature of the linkage type.
DUB reactivity: The potential utility of (K6-only)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 glutathione-derivatised matrices. This may have implications for their successful use in some applications, for example, if they are to be used in concert with GST-tagged proteins requiring subsequent affinity isolation with glutathione-based matrices. Such methods of detection or isolation should be avoided wherever possible in order to avoid complication in interpretation of results obtained.
Figure 1: Coomassie stained SDS-PAGE gel showing ubiquitinylation of substrate protein (X) with (K6-only) Ub to give (K6-only)Ubn-ubiquitinylated conjugates (Z; Prod. No. BML-UW0615)

BML-UW0615 WB assay
Figure 3: Western Blot showing (K6-only)Ubn-ubiquitinylated substrate (Prod. No. BML-UW0615) incubated with (+) or without (-) USP2cd (Prod. No. BML-UW9850), detected with PAb to ubiquitin-protein conjugates.
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BML-UW0615 SDS-PAGE BML-UW0615 WB 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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