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33959
Branched Ubiquitin Antibody Sampler Kit

Branched Ubiquitin Antibody Sampler Kit #33959

Western Blotting Image 1

Western blot analysis of 293 and HeLa cells, untreated or treated with the 26S proteasome inhibitor MG132 (50 µM, 90 minutes), using Ubiquitin (P4D1) Mouse mAb.

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Western Blotting Image 2

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).

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Western Blotting Image 3

Western blot analysis of recombinant monoubiquitin (MonoUb), linear unbranched ubiquitin chain (Ub2-7), K48-linked polyubiquitin (K48-Ub2-7) and K63-linked polyubiquitin (K63-Ub2-7), using K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (upper) and Ubiquitin (P4D1) Mouse mAb #3936 (lower). Ubiquitin chains range from 2 to 7 in length.

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Western Blotting Image 4

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IHC-P (paraffin) Image 5

Immunohistochemical analysis of paraffin-embedded breast carcinoma showing nuclear and cytoplasmic ubiquitin localization, using Ubiquitin (P4D1) Mouse mAb.

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Western Blotting Image 6

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).

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Western Blotting Image 7

Western blot analysis of six distinct recombinant polyubiquitin chains using K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (upper) and Ubiquitin (P4D1) Mouse mAb #3936 (lower).

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Western Blotting Image 8

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).

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Western Blotting Image 9

Western blot analysis of H1703 cells, untreated or treated with MG132 as indicated, using K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb.

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Western Blotting Image 10

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

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Western Blotting Image 11

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.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Ubiquitin (P4D1) Mouse mAb 3936 20 µl
  • WB
  • IHC
All Mouse IgG1
K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb 5621 20 µl
  • WB
All Rabbit IgG
K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb 8081 20 µl
  • WB
All Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Branched Ubiquitin Antibody Sampler Kit provides an economical means of detecting total and common branch specific forms of ubiquitin. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Ubiquitin (P4D1) Mouse mAb detects ubiquitin, polyubiquitin and ubiquitinated proteins. This antibody may cross-react with recombinant NEDD8. Linkage specific antibodies K48-linkage Specific Polyubiquitin (D9D5C6) Rabbit mAb and K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb detects polyubiquitin chains formed by Lys48 and Lys63 residue linkages, respectively. These antibodies do not react with monoubiquitin or polyubiquitin chains formed by specific linkage to different lysine residues.

Ubiquitin (P4D1) Mouse mAb is produced by immunizing animals with a recombinant protein expression amino acids 1-76 of full length bovine ubiquitin. Branch specific monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residus surrounding the Lys48 and Lys63 branches of the human diubiquitin chain.

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 polyubiquinated at specific lysine residues display a tendency to be targeted for different processes; K48-linked polyubiquitin chains mainly target proteins for proteasomal degradation while K63-linked polyubiquitin regulates protein function, subcellular localization, or protein-protein interactions (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, 7151-60.
  2. Hochstrasser, M. (2000) Nat Cell Biol 2, E153-7.
  3. Hochstrasser, M. (2000) Science 289, 563-4.
  4. Bernardi, R. et al. (2000) Oncogene 19, 2447-54.
  5. Aberle, H. et al. (1997) EMBO J 16, 3797-804.
  6. Salomoni, P. and Pandolfi, P.P. (2002) Nat Cell Biol 4, E152-3.
  7. Jesenberger, V. and Jentsch, S. (2002) Nat Rev Mol Cell Biol 3, 112-21.
  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.
Entrez-Gene Id
6233 , 7311 , 7314 , 7316
Swiss-Prot Acc.
P62979 , P62987 , P0CG47 , P0CG48
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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