K63-linkage Specific Polyubiqu

5621:

Western Blotting

K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb detects polyubiquitin chains formed by Lys63 residue linkage. It does not react with monoubiquitin or polyubiquitin chains formed by linkage to a different lysine residue.

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the Lys63 branch of the human diubiquitin chain.

Western Blotting

Western blot analysis of extracts from HeLa cells, untreated or treated with the proteasome inhibitor MG132 (10 µM for 6 hours), using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb and Ubiquitin Antibody #3933 (lower).

Western Blotting

Western blot analysis of seven distinct recombinant polyubiquitin chains (300 ng each) using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb (upper) and Ubiquitin Antibody #3933 (lower).

Western Blotting

Western blot analysis comparing the titration of recombinant monoubiquitin, K48-linked polyubiquitin and K63-linked polyubiquitin using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb (upper) and Ubiquitin Antibody #3933 (lower).

Western Blotting

Western blot analysis of various cell lines using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb.

Western Blotting

Western blot analysis of extracts from HeLa cells using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb, untreated or following antibody pre-incubation with either K63 ubiquitinylated branched peptide to block the signal or a linear peptide surrounding K63 of ubiquitin that cannot block the signal.

Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH₂ of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25a and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7).

Substrate proteins are linked to ubiquitin using seven distinct ubiquitin lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63). Formation of a polyubiquitin chain occurs when a lysine residue of ubiquitin is linked to the carboxy-terminal glycine of another ubiquitin. Proteins polyubiquitinated at specific lysine residues display a tendency to be targeted for different processes (8). K63-linked polyubiquitin chains exert nonproteolytic functions in vivo, such as protein trafficking, kinase/phosphatase activation, and DNA damage control, all of which might be important in regulation of cancer survival and development (9,10).

1. Ciechanover, A. (1998) EMBO J. 17, 7151-7160.
2. Hochstrasser, M. (2000) Nat. Cell Biol. 2, E153-E157.
3. Hochstrasser, M. (2000) Science 289, 563-564.
4. Bernardi, R. et al. (2000) Oncogene 19, 2447-2454.
5. Aberle, H. et al. (1997) EMBO J. 16, 3797-3804.
6. Salomoni, P. and Pandolfi, P.P. (2002) Nat. Cell Biol. 4, E152-E153.
7. Jesenberger, V. and Jentsch, S. (2002) Nat. Rev. Mol. Cell Biol. 3, 112-121.
8. Komander, D. (2009) Biochem Soc Trans 37, 937-53.
9. Chen, Z.J. and Sun, L.J. (2009) Mol Cell 33, 275-86.
10. Yang, W.L. et al. (2010) Oncogene 29, 4493-503.

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• 7071 Phototope^® -HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide
• 4289 K48-linkage Specific Polyubiquitin Antibody
• 3933 Ubiquitin Antibody
• 3936 Ubiquitin (P4D1) Mouse mAb

For Research Use Only. Not For Use In Diagnostic Procedures.