Echelon’s Class III PI3K ELISA Kit is a competitive, 96-well ELISA assay designed to measure the activity of Class III Phosphoinositide 3-Kinases (PI3K) through the detection of its product, PtdIns(3)P (PI(3)P).
Assay Design
Echelon’s Class III PI3K Activity Assay is a competitive ELISA, where the signal is inversely proportional to the amount of PI(3)P generated. After the PI3-K reaction is complete and quenched, the reaction products are diluted and added to a microplate coated with PI(3)P. The PI(3)P competes with the reaction product for binding to a PI(3)P-specific detector protein. The amount of bound PI(3)P detector is measured at 450 nm absorbance. This assay is specific to PI(3)P, allowing for the detection of as little as 1 pmol of PI(3)P in a 100 μL sample, corresponding to a sensitivity of 10 nM.
Product Background
Phosphoinositide 3-Kinases (PI3-K) are categorized into three classes based on structural homology and substrate preference. Class III PI3-K specifically utilizes phosphatidylinositol (PI) as a substrate to generate PI(3)P. While Class I PI3-Ks are well-known for their roles in cell growth and division, the functions of Class II and III PI3-Ks are still being explored. Traditional methods for measuring PI3-K activity typically involved the use of radiolabeled 32P for phosphorylation of a phosphoinositide substrate, followed by radioactive product extraction and separation via thin-layer chromatography or HPLC. Echelon’s assay provides a non-radioactive alternative, offering a straightforward method to assess PI3-K activity using recombinant or immunoprecipitated enzyme in a conventional ELISA format, eliminating the need for radioactive materials or complex separation techniques.
Product Keywords:PI3K, PI3 Kinase, PI3 K, PI3-K, PI3P
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