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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

Click Here for Detailed Methods

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)

Author	Title	Journal	Year	Article Link
Afrin, Sadia et al.	Sim******* modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradation	Pharmacological Research	2021	ISSN 1096-1186
Tumova, Lucie et al.	The ubiquitin-proteasome system does not regulate the degradation of porcine β-microseminoprotein during sperm capacitation	International Journal of Molecular Sciences	2020	ISSN 1422-0067
Gu, Chunming et al.	Identification of berberine as a novel drug for the treatment of multiple myeloma via targeting UHRF1	BMC Biology	2020	ISSN 1741-7007
Chu, Yi et al.	O-GlcNAcylation of SIX1 enhances its stability and promotes hepatocellular carcinoma proliferation	Theranostics	2020	ISSN 1838-7640
Uchiyama, Makoto et al.	O2-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel Responses	Current Biology	2020	ISSN 1879-0445
Sen, Arpita et al.	The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization	Journal of Cell Science	2020	ISSN 1477-9137
Park, Jin Suk et al.	Mechanical regulation of glycolysis via cytoskeleton architecture	Nature	2020	ISSN 1476-4687

Author

Title

Journal

Year

Article Link

Afrin, Sadia et al.

Sim******* modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradation

Pharmacological Research

2021

ISSN 1096-1186

Tumova, Lucie et al.

The ubiquitin-proteasome system does not regulate the degradation of porcine β-microseminoprotein during sperm capacitation

International Journal of Molecular Sciences

2020

ISSN 1422-0067

Gu, Chunming et al.

Identification of berberine as a novel drug for the treatment of multiple myeloma via targeting UHRF1

BMC Biology

2020

ISSN 1741-7007

Chu, Yi et al.

O-GlcNAcylation of SIX1 enhances its stability and promotes hepatocellular carcinoma proliferation

Theranostics

2020

ISSN 1838-7640

Uchiyama, Makoto et al.

O2-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel Responses

Current Biology

2020

ISSN 1879-0445

Sen, Arpita et al.

The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization

Journal of Cell Science

2020

ISSN 1477-9137

Park, Jin Suk et al.

Mechanical regulation of glycolysis via cytoskeleton architecture

Nature

2020

ISSN 1476-4687

细胞骨架/信号导引头™ 泛素化检测试剂盒（10次检测）/10次检测/BK161-S