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([K6-only]Ub)n-ubiquitinylated substrate
Product Specification
MW: ~50-250kDa
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: P62988
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: 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.
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. |