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Auto-ubiquitinylation kit

BML-UW0970

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Well-validated test to assess ubiquitin E3 ligase activity

Qualitative assessment of an Ub E3 ligase enzyme’s activity through its ability to auto-ubiquitinylate.
Testing of proteins for auto-ubiquitinylation activity allowing their identification as putative ubiquitin E3 ligases.
Ubiquitinylation of substrate proteins (user provided) specific to a particular ubiquitin E3 ligase.

This E3 ligase auto-ubiquitinylation kit enables proteins to be tested for ubiquitin E3 ligase activity through assessment of their ability to under auto-ubiquitinylation. Utilizing the first three steps in the ubiquitin cascade the kit facilitates ubiquitinylation of known or putative E3 ligase enzymes followed by Western blot analysis using the highly sensitive reagents provided or using antibodies to the specific protein of interest (user supplied). A high integrity ubiquitin E3 ligase enzyme is also provided for use as a positive control.

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Auto-ubiquitinylation kit Western blot — Western blot analysis of control Ub E3 ligase Hdm2 RING domain auto-ubiquitinylation assays. Auto-ubiquitinylation assays set-up and run as described in “Assay protocol”. Ubiquitinylated E3 ligase species were detected by Western blotting as described in “Analysis by western blotting”, using the provided ubiquitin antibody (Prod. No. ADI-SPA-203) at a dilution of 1:1,000. Results demonstrate auto-ubiquitinylation of the control Hdm2 RING domain ligase under the given assay conditions. (E2-Ub (thioester linked) is removed in reducing condition. Left – non-reducing; Right – reducing condition.)
Lane 1: Pre-stained Protein Standard. Lane 2: Hdm2-Ub, (+) control, (non-reduced). Lane 3: Hdm2, (-) control, (no Mg-ATP), (non-reduced). Lane 4: Hdm2-Ub, (+) control, (reduced). Lane 5: Hdm2, (-) control, (no Mg-ATP), (reduced). Lane 6: Hdm2-Ub, (+) control, (reduced). Lane 7: Hdm2, (-) control (no ubiquitin), (reduced).

Product Details

Application Notes	For assessment of protein Ub E3 ligase activity.
Contents	20X Ubiquitin Activating Enzyme Solution (E1), 25µl 20X Ubiquitin Conjugating Enzyme Solution (E2), 25µl 10X Ubiquitin Solution (Ub), 50µl 20X Control Ubiquitin Ligase Solution (E3), 25µl 20X Mg-ATP Solution, 25µl 10X Ub E3 ligase Buffer, 50µl Ubiquitin antibody solution, 25µl
Quantity	Sufficient for 10 auto-ubiquitinylation assays.

Handling & Storage

Handling	Avoid freeze/thaw cycles.
Short Term Storage	-80°C
Long Term Storage	-80°C
Shipping	Dry Ice

Regulatory Status	RUO – Research Use Only

The tRNA-GCN2-FBXO22-axis-mediated mTOR ubiquitination senses amino acid insufficiency: M.-K. Ge, et al.; Cell Metab. 35, 2216 (2023), Abstract
Overexpression of ARM repeat/U-box containing E3 ligase, PUB2 positively regulates growth and oxidative stress response in Arabidopsis: L.K. Saini, et al.; Biochem. J. 480, 555 (2023), Abstract
Expression quantitative trait loci mapping identified PtrXB38 as a key hub gene in adventitious root development in Populus: T. Yao, et al.; New Phytol. , (2023), Abstract
p21-activated kinase 4 controls the aggregation of α-synuclein by reducing the monomeric and aggregated forms of α-synuclein: involvement of the E3 ubiquitin ligase NEDD4-1: S.Y. Won, et al.; Cell Death Dis. 13, 575 (2022), Abstract
Arabidopsis thaliana Ubiquitin-Associated Protein 1 (AtUAP1) Interacts with Arabidopsis thaliana Ring Zinc Finger 1 (AtRZF1) to Negatively Regulate Dehydration Response: J.H. Min, et al.; Plant Cell Physiol. 62, 1044 (2021), Abstract
PLPP/CIN-mediated Mdm2 dephosphorylation increases seizure susceptibility via abrogating PSD95 ubiquitination: J.E. Kim, et al.; Exp. Neurol. 331, 113383 (2020), Abstract
Genome-wide identification of genes encoding putative secreted E3 ubiquitin ligases and functional characterization of PbRING1 in the biotrophic protist Plasmodiophora brassicae: F. Yu, et al.; Curr. Genet. 65, 1355 (2019), Abstract
PAMP-responsive ATL gene StRFP1 and its orthologue NbATL60 positively regulate Phytophthora infestans resistance in potato and Nicotiana benthamiana: C. Zhong, et al.; Plant Sci. 270, 47 (2018), Abstract
Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification: M. Tsuru, et al.; PLoS One 13, e0196204 (2018), Abstract — Full Text
The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis: J.Y. Zhu, et al.; Mol. Cell 66, 648 (2017), Abstract — Full Text

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Auto-ubiquitinylation kit

BML-UW0970

Product Details

Handling & Storage

Datasheet, Manuals, SDS & CofA

Manuals And Inserts

Certificate of Analysis

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