PI3K activity was determined using a commercially available PI3K ELISA kit (Echelon Biosciences Inc) according to the manufacturer’s instructions. Briefly, after drug treatment, cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed in 500-μL ice-cold lysis buffer (137-mM NaCl, 20-mM Tris–HCl [pH 7.4], 1-mM CaCl2, 1-mM MgCl2, 1-mM Na3VO4, 1% NP-40, and 1-mM PMSF). PI3K was then immunoprecipitated with 5 μL of antibody (anti-p85α, RRID: AB_2714180) and 60 μL of Pierce Protein A/G magnetic beads (Thermo Scientific). PI3K activity in the immunoprecipitates was then assayed by PI3K ELISA according to the manufacturer’s instructions. The spectrophotometric data were obtained using a Synergy 2 Multi-Detection Microplate Reader (Agilent Technologies) at a wavelength of 450 nm. The protein concentrations of cellular lysates were determined by Bradford assay as described earlier. The activity of PI3K was corrected for protein content.
Pi3k elisa kit
Manufactured by Echelon Biosciences
The PI3K ELISA kit is a laboratory assay designed to detect and quantify the levels of Phosphoinositide 3-kinase (PI3K) in biological samples. PI3K is an important enzyme involved in various cellular processes, and its expression or activity can provide insights into cellular signaling pathways. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) technique to measure PI3K levels in a reliable and reproducible manner.
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2 protocols using pi3k elisa kit
1
Quantifying PI3K Activity by ELISA
2
PI3K Activity Determination by ELISA
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PI3K activity was determined using a commercially available PI3K ELISA kit (Echelon Biosciences Inc., Salt Lake City, UT) according to the manufacturer's instructions. Briefly, after drug treatment, cells were washed in ice-cold PBS and lysed in 500 μl ice-cold lysis buffer (137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM CaCl2, 1 mM MgCl2, 1 mM Na3VO4 , 1% NP-40 and 1 mM PMSF). PI3K was then immunoprecipitated with 5 μl of antibody (anti-p85α, MilliporeSigma, Burlington, MA, USA) and 60 μl of Pierce Protein A/G magnetic beads (Thermo Scientific). PI3K activity in the immunoprecipitates was then assayed by PI3K ELISA according to the manufacturer's instructions. The spectrophotometric data were obtained using a Synergy 2 Multi-Detection Microplate Reader (Agilent Technologies, Santa Clara, CA, USA) at a wavelength of 450 nm. The protein concentrations of cellular lysates were determined by Bradford assay as described above.
The activity of PI3K was corrected for protein content.
The activity of PI3K was corrected for protein content.
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