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PDP - Template Name: ELISA Kit
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PathScan® Phospho-Ubiquitin (Ser65) Sandwich ELISA Kit #28384

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  • ELISA

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    Supporting Data

    REACTIVITY H
    Application Key:
    • ELISA-ELISA 
    Species Cross-Reactivity Key:
    • H-Human 

    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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