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PathScan® Phospho-Ubiquitin (Ser65) Sandwich ELISA Kit #28384

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  • ELISA
ELISA Image 1: PathScan® Phospho-Ubiquitin (Ser65) Sandwich ELISA Kit
Figure 1. Treatment of 293 cells with valinomycin stimulates phosphorylation of ubiquitin at Ser65. The relationship between lysate protein concentration from untreated and valinomycin-treated 293 cells and the absorbance at 450 nm using the PathScan® Phospho-Ubiquitin (Ser65) Sandwich ELISA Kit #28384 is shown in the left figure. The corresponding western blots using phospho-ubiquitin (Ser65) antibody (left panel) and ubiquitin antibody (right panel) are shown in the right figure. 293 cells were treated with valinomycin (1 µM) for 24 hours at 37°C and then lysed.

To Purchase # 28384**

Important Ordering Details

Custom Ordering Details:

If kit quantities from the same lot are needed in unlisted sizes, contact us for processing time and pricing.

Looking for this ELISA kit in a 384-well format? Inquire for availability, processing time, and pricing.

Supporting Data

REACTIVITY H
Application Key:
  • ELISA-ELISA 
Species Cross-Reactivity Key:
  • H-Human 
  • Product Includes
  • Related Products
Product IncludesVolumeSolution Color
Phospho-Ubiquitin (Ser65) Rabbit mAb Coated Microwells #4281596 tests
Ubiquitin Mouse Detection mAb #607371 eaGreen (Lyophilized)
Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated) #133041 eaRed (Lyophilized)
Detection Antibody Diluent #1333911 mlGreen
HRP Diluent #1351511 mlRed
TMB Substrate #700411 ml
STOP Solution #700211 ml
Sealing Tape #545032 ea
ELISA Wash Buffer (20X) #980125 ml
ELISA Sample Diluent #1108325 mlBlue
Cell Lysis Buffer (10X) #980315 ml

Kit contents scale proportionally with size, except sealing tape.
Example: The V1 kit contains 5X the listed quantities above, but will exclude the sealing tape.

The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.

Product Information

Product Description

The PathScan® Phospho-Ubiquitin (Ser65) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of ubiquitin protein phosphorylated at Ser65. A phospho-ubiquitin (Ser65) rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-ubiquitin (Ser65) protein is captured by the coated antibody. Following extensive washing, a ubiquitin mouse detection antibody is added to detect the captured phospho-ubiquitin (Ser65) protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of ubiquitin protein phosphorylated at Ser65.

*Antibodies in this kit are custom formulations specific to kit.

Protocol

Specificity / Sensitivity

The PathScan® Phospho-Ubiquitin (Ser65) Sandwich ELISA Kit detects endogenous levels of ubiquitin protein phosphorylated at Ser65. The kit sensitivity is shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

Human

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).
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