K-400

Material Data Sheet

UbiCREST Deubiquitinase Enzyme Kit

Price: $439.00
Catalog #: K-400

UbiCREST Deubiquitinase Enzyme Kit

Price: 
$439
Quantity: 
1 Kit
Data Sheet: 

UbiCREST (Ubiquitin Chain Restriction Analysis) kits, developed in the Komander lab and under exclusive license to Boston Biochem, Inc., are designed to analyze the ubiquitin linkages present in free polyubiquitin chains or polyubiquitin chains conjugated to a substrate protein. The UbiCREST approach utilizes the linkage specificity of the ovarian tumor domain (OTU) class of deubiquitinases (DUBs) to characterize the linkages present in a sample containing polyubiquitin. This kit provides DUBs that are useful for in vitro analysis of user supplied polyubiquitinated protein samples. The samples may be generated by in vitro ubiquitination reactions, or obtained from cell lysates or other complex mixtures following immunoprecipitation or other means of purification.

Covalent attachment of ubiquitin to target proteins is one of the most versatile and intriguing post-translational modifications observed in eukaryotic organisms. Because polyubiquitin chains may be generated by connecting adjacent ubiquitin molecules in one of eight different orientations, a large number of varied architectures may be generated. Polyubiquitination of proteins directs them to different cellular fates such as translocation to different areas within the cell, assembly into multi-subunit complexes, or degradation via the proteasome, depending on the type of linkage used in the polyubiquitin chain. Recent studies have begun to define the roles of these different polyubiquitin signals in physiology and disease, and it is obvious that identifying and understanding the uses of the various linkages will be important in understanding and treating human disease. Polyubiquitin chains are typically formed via the E1, E2, E3 enzyme conjugation cascade where the C-terminal glycine of one ubiquitin is linked to the ε-amino group of a lysine on another ubiquitin; this linkage is referred to as an isopeptide bond. Polyubiquitin chains of all possible linkages including K6-, K11-, K27-, K29-, K33-, K48-, K63-, and Met1-linked (or "linear") have been detected in vivo.

Product Information

Western Blot Data: 
Western Blot Data: 
Supplied: 
Kit contains reagents sufficient for 5 UbiCREST analysis experiments.  Kit components include: 10X OTUB1, 10X AMSH, 10X OTUD3 catalytic domain, 10X Trabid, 10X Cezanne catalytic domain, 10X Yod1, 10X Otulin, 10X USP2 catalytic domain, and 10X DUB Reaction Buffer.
Recommended Assay Protocol: 
UbiCREST Analysis provides a rapid and economical means of identifying the types of linkages present in polyubiquitin chains. Briefly, the method consists of purifying a polyubiquitinated substrate, treating it with a panel of linkage-specific deubiquitinases (8 reactions in parallel), then analyzing the data to determine which of the DUBs were active against the substrate. Data output may consist of silver stain or Western Blot visualization of liberated ubiquitin and polyubiquitin chains ("Primary analysis"), or Western Blot analysis of the Target Protein ("Secondary analysis"). While the methods are straightforward, it is often useful to generate two readouts (ubiquitin-based and Target-based) in order to obtain a more comprehensive picture of the linkage(s) present in a ubiquitinated substrate. Both types of data may be generated from a single experiment.
Storage: 

May be stored at -80 °C.*  Avoid multiple freeze-thaw cycles.

Literature

References: 

Fushman, D. and K.D. Wilkinson (2001) F1000 Biol Reports 3:26

Hospenthal, M. K. et al. (2015) Nat. Protocols 10: 349

Komander, D. (2009) Biochem Soc Trans. 37: 937

Komander, D. and M. Rape (2012) Ann. Rev. of Biochem. 81: 203.

Mevissen, T.E. et al. (2013) Cell 154: 169.

Peng, J. et al. (2003) Nat Biotechnol 8: 921.