Revision 5
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.
Applications:

WB, IP

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

Source/Isotype:

Rabbit IgG

UniProt ID:

#P62987, #P0CG48, #P0CG47, #P62979

Entrez-Gene Id:

7311, 7316, 7314, 6233

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100

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.

Specificity / Sensitivity

Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb recognizes endogenous levels of Ubiquitin protein only when phosphorylated at Ser65.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology

Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser65 of human Ubiquitin protein.

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).

Ubiquitin is phosphorylated at Ser65 by PINK1, leading to activation of the E3 ubiquitin ligase Parkin (8, 9). PINK1 accumulates on depolarized mitochondria, resulting in phosphorylation of ubiquitin and activation of Parkin, which then triggers the mitophagy pathway to clear damaged mitochondria. Loss-of-function mutations in PINK1 and Parkin result in early onset Parkinson's disease (10, 11).

  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. Kane, L.A. et al. (2014) J Cell Biol 205, 143-53.
  9. Koyano, F. et al. (2014) Nature 510, 162-6.
  10. Kitada, T. et al. (1998) Nature 392, 605-8.
  11. Valente, E.M. et al. (2004) Science 304, 1158-60.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered 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 5
#62802

Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb

Western Blotting Image 1: Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb Expand Image
Western blot analysis of extracts from PC-3 cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 30 μM, 6 hr; +), using Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb (upper), PINK1 (D8G3) Rabbit mAb #6946 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 2: Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb Expand Image
Western blot analysis of extracts from PC-3 cells treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 30 μM, 6 hr ). Lysates were treated with (+) or without (-) Lambda Phosphatase (λ-Phosphatase) and Calf Intestinal Phosphatase (CIP). Western blot was performed using Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb Expand Image
Immunoprecipitation of phospho-ubiquitin (Ser65) from extracts of PC-3 cells treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 30 μM, 6 hr). Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb. Western blot was performed using Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb. Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.