Cytoskeleton/Signal-Seeker™ Ubiquitination Detection Kit (30 assay)/30 assays/BK161188bio精品生物—专注于实验室精品爆款的电商平台

Details

The Signal-Seeker™ line of produts have been developed to allow simple analysis of key regulatory protein modifications by specialists and non-specialists alike. The comprehensive Signal-Seeker™ kits provide an affinity bead system to isolate and enrich modified proteins from any given cell or tissue lysate. The enriched protein population is then analyzed by standard western blot procedures using a primary antibody to the target protein.

Product Uses Include

Investigate transient regulatory mechanisms
Measure signalling events of multiple pathway member proteins
Discover new modifications of your protein of interest
Gain insight into regulatory mechanisms
Measure endogenous or transiently expressed protein signalling events

Validation Data: Ubiquitination Detection Kit White Paper

Kit contents

The Ubiquitination kit contains the following components:

Lysis and protein quantitation step

IP and pre-clear step

Wash step

Elution step

Western step

BlastR™ Lysis Buffer

BlastR™ Dilution Buffer

BlastR™ Filters

Protease Inhibitor Cocktail

De-Ubiquitinase inhibitor Cocktail

Precision Red™ Protein Assay Reagent

Ubiquitination Affinity Beads

Ubiquitination Control Beads

BlastR™ Wash Buffer

Spin Columns

Bead Elution Buffer

Chemiluminescent Reagent A

Chemiluminescent Reagent B

Anti-ubiquitin-HRP antibody

Example results

There are many applications for these kits (see next section below), here we describe an interesting application for Rac1 protein:

Application 1: Investigate highly transient regulation events

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. 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.

Other experiments that could be attempted in this area of research include:

• Pharmacological investigation of ubiquitinases or de-ubiquitinases involved in Rac1 mono- and poly-ubiquitination

• Investigation of the relationship between Rac1 activation and ubiquitination under a variety of different growth factors or drug treatments.

• Examine the interaction of ubiquitinated Rac1 with effectors.

• Examine crosstalk between ubiquitinated Rac1 and other regulatory PTMs of Rac1.

For more information contact: signalseeker@cytoskeleton.com

Associated Products:

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

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

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

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

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

About

Click on the pdf icon below to download the manual

Citations

For the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click here

Faqs

Visit our Signal-Seeker™ Tech Tips and FAQs page for technical tips and frequently asked questions regarding this and other Signal-Seeker™ products click here

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com

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Cytoskeleton/Signal-Seeker™ Ubiquitination Detection Kit (30 assay)/30 assays/BK161