([K11-only]Ub)n-ubiquitinylated substrate

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. (K⁶-only)Ub_n-ubiquitinylated substrate, Prod. No. BML-UW0615)
3. Investigation of polyubiquitin chain recognition by and interaction with ubiquitin binding proteins.

Liquid. In 50mM TRIS, pH 7.5, containing 150mM NaCl and 1mM DTT.

~50-250kDa

≥95% (SDS-PAGE)

High molecular weight conjugates were separated from free ubiquitin by size exclusion chromatography.

SDS-PAGE: Multiple high molecular weight bands are observed for (K¹¹-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-TLTGK(G-G)TITLEVEPSDTIENVK-OH) was identified, confirming the polyubiquitinylated status of the conjugates and the nature of the linkage type.
DUB reactivity: The potential utility of (K¹¹-only)Ub_n-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).

Protein components produced in E. coli. Synthesized enzymatically in vitro. (K¹¹-only)Ub_n-ubiquitinylated substrate protein was produced using K¹¹-only mutant ubiquitin.

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.

P0CG47 (UBB), P0CG48 (UBC), P62979 (RPS27A), P62987 (UBA52)