Auto-ubiquitinylation kit



BML-UW0970-0001	1 Kit	201.00 USD

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.

Figure: Western blot analysis of control Ub E3 ligase Hdm2 RING domain auto-ubiquitinylation assays. Auto-ubiquitinylation assays set-up and run as described in the manual 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. BML-KW9510) at a dilution of 1:1000.

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Product Specification

Quantity:	Sufficient for 10 auto-ubiquitinylation assays.

Kit/Set Contains:	20X Ubiquitin Activating Enzyme Solution (E1) (Prod. No. BML-KW9410) 25µL 20X Ubiquitin Conjugating Enzyme Solution (E2) (Prod. No. BML-KW9050) 25µL 10X Ubiquitin Solution (Ub) (Prod. No. BML-KW8795) 25µL 20X Control Ubiquitin Ligase Solution (E3) (Prod. No. BML-KW0200) 25µL 20X Mg-ATP Solution (Prod. No. BML-KW9805) 25µL 10X Ub E3 ligase Buffer (Prod. No. KW0965) 50µL Ubiquitin antibody solution (Prod. No. BML-KW9510) 25µL

Application:	For assessment of protein Ub E3 ligase activity.

Short Term Storage:	-80°C

Long Term Storage:	-80°C

Handling:	Avoid freeze/thaw cycles.

Miscellaneous/General:	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.

General Literature References

The UBXN1 protein associates with Autoubiquitinated forms of the BRCA1 tumor suppressor and inhibits its enzymatic function: F. Wu-Baer, et al.; Mol. Cell Biol. 30, 2787 (2010), Abstract;

Autoubiquitination of BCA2 RING E3 ligase regulates its own stability and affects cell migration: Y. Amemiya, et al.; Mol. Cancer Res. 6, 1385 (2008), Abstract;

Mechanisms underlying ubiquitination: C.M. Pickart; Annu. Rev. Biochem. 70, 503 (2001), Abstract;

The ubiquitin-proteasome system and endocytosis: G.J. Strous & R. Govers; J. Cell Sci. 112 (Pt 10), 1417 (1999), Abstract;

The ubiquitin system: A. Hershko & A. Ciechanover; Annu. Rev. Biochem. 67, 425 (1998), Abstract;

Pathways of ubiquitin conjugation: A.L. Haas & T.J. Siepmann; FASEB J. 11, 1257 (1997), Abstract;