Revision 1

#33959Store at -20C

1 Kit

(3 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Ubiquitin (P4D1) Mouse mAb 3936 20 µl Mouse IgG1
K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb 5621 20 µl Rabbit IgG
K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb 8081 20 µl Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

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.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #33959

    Branched Ubiquitin Antibody Sampler Kit

    Branched Ubiquitin Antibody Sampler Kit: Image 1 Expand Image
    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.
    Branched Ubiquitin Antibody Sampler Kit: Image 2 Expand Image
    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).
    Branched Ubiquitin Antibody Sampler Kit: Image 3 Expand Image
    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.
    Branched Ubiquitin Antibody Sampler Kit: Image 4 Expand Image
    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.
    Branched Ubiquitin Antibody Sampler Kit: Image 5 Expand Image
    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.
    Branched Ubiquitin Antibody Sampler Kit: Image 6 Expand Image
    Immunohistochemical analysis of paraffin-embedded breast carcinoma showing nuclear and cytoplasmic ubiquitin localization, using Ubiquitin (P4D1) Mouse mAb.
    Branched Ubiquitin Antibody Sampler Kit: Image 7 Expand Image
    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).
    Branched Ubiquitin Antibody Sampler Kit: Image 8 Expand Image
    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).
    Branched Ubiquitin Antibody Sampler Kit: Image 9 Expand Image
    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).
    Branched Ubiquitin Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of H1703 cells, untreated or treated with MG132 as indicated, using K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb.
    Branched Ubiquitin Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of various cell lines using K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb.
    Branched Ubiquitin Antibody Sampler Kit: Image 12 Expand Image
    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.