"My laboratory has published two recent manuscripts reporting the use of FLAG-K63 and FLAG K48 TUBE reagents. We used these reagents to study the ubiquitination of the nonstructural 2 transmembrane protein of hepatitis C virus (HCV) by the cellular E3 ligase MARCH8. By being quite selective and easy to handle, these reagents helped us characterize the specific ubiquitin modification."
Dr. Shirit Einav, Associate Professor, Department of Microbiology and Immunology, Stanford University of Medicine
FLAG® Anti-K63 TUBE is an ideal reagent for efficient isolation and enrichment of K63-polyubiquitinated proteins from cell & tissue extracts or in vitro synthesized mixtures. Based on a peptide engineered by Sims et al., it consists of multiple ubiquitin interaction motifs (UIMs) joined by a rigid, helical linker that spaces the UIMs for selective binding to extended K63-linked polyubiquitin chains. The result is a peptide the exhibits high affinity binding to K63-linked polyubiquitin together with 1000 to 10,000-fold selectivity over K48- and K11- linkages. Expression of this peptide in vitro inhibits K63-linked polyubiquitin-dependent processes and protects K63-linked polyubiquitin chains from degradation. Combining this peptide with a FLAG epitope tag generates, for the first time, a powerful affinity reagent suitable for isolation, purification and characterization of proteins modified by K63-linked polyubiquitin. Flag K63-TUBE 1 allows isolation of K63-linked polyubiquitin without the need for overexpression of ubiquitin mutants, tagged ubiquitins or the inclusion of DUB inhibitors any of which could alter cellular physiology. Isolated ubiquitinated proteins can be characterized by Western blot, mass spec or further biochemical analysis. (FLAG is a registered trademark of Sigma-Aldrich Corporation LLC).
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|Molecular Weight||9062.7 (calculated)|
|Formulation||1 mg/mL (110uM) in PBS, pH 7.2|
|Recommended Concentration||50-500 nM|
- Isolation and enrichment of K63-polyubiquitinated proteins from cell and tissue extracts
- Isolation of ubiquitinated proteins for proteomic studies
- Nanomolar dissociation constant (Kd) for K63-chains
- 1000 to 10,000-fold preference for K63 chains over K48- or K11- chains
- Overexpression of epitope-tagged ubiquitin for pull downs not necessary
- Compatible with FLAG® technology, providing flexibility and specificity
 Sims, JJ, Cooper, EM, Scavone, F, Kane, LA, Youle, RJ, Boeke, JD, and Cohen, RE. (2012).“Ubiquitin sensor peptides reveal localization and linkage-type dependence of cellular polyubiquitin signaling.” Nature Methods, 9(3):303-9.
 Hjerpe, R, Aillet, F, Lopitz-Otsoa F, Lang, V, England P, and Rodriguez, MS. (2009). “Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities.” EMBO Rep., 10(11):1250-8.