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Details

As part of the Signal-Seeker™ product line, Ubiquitin affinity beads have been optimized in order to detect endogenous levels of mono- and poly-ubiqutinated proteins, which often represent <1% of the target protein. UBA01-Beads can enrich all ubiquitinated species with a high affinity, including mono-, multi- and polyubiquitin chains (see Application 1 below). The affinity moeities have been cross-linked to beads for low leaching and cleaner detection of the protein of interest. A comprehensive Signal-Seeker™ Ubiquitination Detection Kit is also available (BK161) and is recommended for first time users.

Validation Data: Ubiquitination Affinity Beads White Paper

Each lot of affinity-bead is quality controlled to provide high batch to batch consistency, see COA documents.

Validated Applications

Application 1: Investigate mono- and poly-ubiquitination

The activity of the Rho family of proteins, including Rac1, is known to be regulated, in part, through ubiquitination (also termed ubiquitylation) events that can lead to signaling pathway regulation via degradation and/or localization of the modified protein (Visvikis, O. et al. 2010. Biol. Cell 102: 377-389. Nethe, M., & Hordijik, P.2010. J. Cell Sci. 123: 4011-4018). In many cases, the GTP-bound active form of Rac1 is the preferred substrate for ubiquitination. For example, it has been shown that cells treated with the bacterial toxin CNF1 leads to constitutive activation of Rac1 and subsequent mono- and poly-ubiquitination (Pop, M. et al. 2004. J. Biol.Chem. 279: 35840-35848).

Using ubiquitin affinty beads (Cat. # UBA01-beads) as part of the Signal Seeker™ Ubiquitination Detection Kit (Cat # BK161) we examined the ubiquitination of endogenous Rac1 in 3T3 cells treated with CNF1 toxin (Cat # CN04) and found that both mono- and polyubiquitination of Rac1 could be detected from 300 μg of 3T3 cell lysate. The kit offers a user friendly tool to examine mono- and poly-ubiquitination for any target protein.

Figure 1: Swiss 3T3 cells were pre-treated with MG-132, then either untreated or treated with CNF1 (CN04) for 3 hours prior to lysis with BlastR buffer. The BK161 kit was utilized to perform the IP on 300 μg of lysate per condition. Lane 1: 3 μg input untreated lysate, Lane 2: Ubiquitin Affinity beads (UBA01) plus untreated lysate, Lane 3: UBA01 plus CN04 treated lysate, Lane 4: ubiquitin control beads (CUB02) plus untreated lysate, Lane 5: CUB02 plus CN04 treated lysate. Samples were analyzed for Rac1 ubiquitination using an anti-Rac1 antibody.

Amount:Each package contains enough ubiquitin beads for 40 reactions and sufficient control beads for 5 reactions.

For more information contact: signalseeker@cytoskeleton.com

Associated Products:

Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)

Signal-Seeker™ SUMOylation 2/3 Detection Kit (Cat. # BK162)

Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)

Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)

Signal-Seeker™: PTMtrue™ Ubiquitin Antibody (Cat.# AUB01)

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Citations

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细胞骨架/泛素化亲和珠/40反应/UBA01珠