Gamma Secretase Activity Assay
Background of Gamma Secretase
Amyloid precursor protein (APP) is involved in neuronal development, which regulates neuronal protein trafficking, neurite outgrowth, synaptogenesis, transmembrane signal transduction, etc. Three proteases participate in the APP cleavage, including α, β, and γ-secretase. The sequential cleavage product of APP is neurotoxic small peptide amyloid β (Aβ). The overexpression and accumulation of the form of Aβ42 peptides in the brain will trigger the further deposition of insoluble fibrillar plaques, which lead to neurodegenerative Alzheimer's disease (AD). Besides β-secretase, γ-secretase is also considered to be associated with AD. The enhanced γ-secretase activity appears to attribute to the formation of Aβ peptide, which is caused by the substitutes Ile for Val718 of APP. This APP mutation is referred to as the "London" mutation. The γ-secretase is now becoming a promising drug target for AD. Developing effective inhibitors of γ-secretase could help us find a cure for AD. Creative Biolabs offers in vitro gamma secretase activity assay services to help our global customers in revealing further mechanisms of AD and developing novel treatments.
Fig.1 Schematic depiction of γ-secretase and the amyloidogenic pathway. (Kanatsu & Tomita, 2016)
Gamma Secretase Activity Assay Services at Creative Biolabs
Gamma secretase activity assay at Creative Biolabs uses a fluorometric reaction to detect the enzymatic activity of the γ-secretase related to the cleavage of APP from cell lysates. Our gamma secretase activity assay process is as follows. First, the intracellular contents of cells are collected from lysed cells. When the γ-secretase cleavages the APP, the fluorescent signal is released and able to be detected by a fluorimeter. The result of the fluorometric reaction is proportional to the level of γ-secretase enzymatic activity.
Another gamma secretase activity assay provided by us is similar to the APP processing assay, which can be utilized to find the γ-secretase inhibitor. It is an in vitro assay established on a stable cell line expressing green fluorescent APP, which includes the γ-secretase cleaving site. The cells are incubated with candidate substances to assess how they affect the APP processing. The nucleus was then labeled with nuclear counterstain. Using image analysis tools, the inhibited cleavage APP will show the fluorescence.
Understanding the mechanisms of APP processing assists researchers to explore the inhibitors of γ-secretase that can subsequently prevent the Aβ formation and aggregation, which facilitates drug development for AD. Creative Biolabs' experienced team offers reliable in vitro gamma secretase activity assays that allow researchers to precisely assess how the drug candidates influence γ-secretase activity in a multiparametric manner. If you have the intention to learn more about APP processing in vitro assays, please don't hesitate to contact us.
- Kanatsu, K.; Tomita, T. Membrane trafficking and proteolytic activity of γ-secretase in Alzheimer's disease. Biological Chemistry. 2016, 397(9): 827-835.