Cytoskeleton蚂蚁淘在线

Details

As part of the Signal-Seeker™ product line, ubiquitination control beads (CUB02) have been developed as highly specific negative control beads for Cytoskeleton"s ubiquitination affinity bead (UBA01-Beads) products. These control beads are far superior to beads alone, because it has mutated versions of the UBDs covalently attached to the control bead matrix. This control is particularly important for ubiquitin IPs as the unmodified version of target proteins often bind non-specifically to all available ubiquitin enrichment affinity matrices. A comprehensive Signal-Seeker™ Ubiquitin Detection Kit (BK161), which includes ubiquitin control beads (CUB02), is available and is recommended for first time users.

Validation Data: Ubiquitination Control 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: Utilization of IgG control beads

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 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.

Importantly, note that the non-specific interaction of unmodified Rac1 occurs with both ubiquitination affinity beads (UBA01) as well as ubiquitination control beads (CUB02) (lanes 2-5); however, the mono- and poly-ubiquitinated Rac1 only binds to ubiquitination affinity beads (lane 3) and not to the ubiquitination control beads (lane 5). This data shows the utility of having appropriate control beads to help users distinguish the unmodified target protein band that is often present when performing ubiquitin IPs, which often occurs with every available ubiquitination enrichment tool.

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. Input: 3 μg input untreated lysate, Ubiquitin Affinity beads (UBA01) plus untreated lysate, UBA01 plus CN04 treated lysate, ubiquitin control beads (CUB02) plus untreated lysate, and CUB02 plus CN04 treated lysate. Samples were analyzed for Rac1 ubiquitination using an anti-Rac1 antibody.

Amount:Each package contains enough ubiquitin control beads for 10 reactions.

For more information contact: signalseeker@cytoskeleton.com

Associated Products:

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

Signal-Seeker™ Ubiquitin Affinity Beads (Cat.# UBA01-beads)

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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Cytoskeleton/Ubiquitination Control Beads/10 reactions/CUB02-beads