PI3K activity was detected by a PI3K activity ELISA kit (Echelon Biosciences Inc., Salt Lake City, UT, USA) according to the manufacturer's instructions. Briefly, PI3K protein (Sigma-Aldrich, St. Louis, USA) was reacted with PI(4,5)P2 as a substrate in a reaction buffer without and with 80 nM of digoxin at 30 ℃ for 2 h. After termination of the reaction, a primary PI(3,4,5)P3 detector was added to the reaction mixture and incubated for 60 min at room temperature. The mixture was transferred to a PI3K ELISA plate, and 60 min later a second detector was added and further incubated at room temperature for 30 min. A tetramethylbenzidine solution was added to the final reaction mixture, and absorbance were measured at 450 nm using a microplate reader. PI(3,4,5)P3 production (pmol/2 h) was regarded as an index of PI3K activity.
Pi3k activity elisa kit
Manufactured by Echelon Biosciences
Sourced in United States
The PI3K activity ELISA kit is a laboratory instrument designed to measure the activity of phosphoinositide 3-kinase (PI3K) in biological samples. It uses an enzyme-linked immunosorbent assay (ELISA) technique to quantify the levels of PI3K activity.
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3 protocols using pi3k activity elisa kit
1
Quantifying PI3K Activity via ELISA
2
Quantifying PI3K Activity by ELISA
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PI3K activity was determined with the PI3K Activity ELISA kit (Echelon Biosciences) according to the manufacturer's instructions [23] (link), [24] (link). For direct functional assessment of PI3K activity, PI3K was isolated by immunoprecipitation using an anti-PI3K antibody (Millipore, #06-195) to the p85 adapter subunit, and the ability of the co-precipitated catalytic p110 catalytic subunit to convert a standard PIP2 to PIP3 in a kinase reaction assessed by measuring the generated PIP3 by the ELISA kit. Each experiment was repeated for three times in duplicates.
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PI3K Activity Measurement Protocol
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The PI3K activity was determined by measuring the level of PI (3, 4, 5) and P3 (PIP3) converted from PI3K substrate PI (4, 5) and P2 (PIP2). A PI3K activity ELISA kit (Echelon Biosciences, Salt Lake City, UT) was used according to the user's instructions.
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