A single molecule TIRFM assay was employed to measure the kinase activity of purified PI3K molecules on a supported lipid bilayer by counting the number of product PIP3 molecules they produce.
This assay utilizes physiological concentrations class I PI3Ka, 1mM ATP, drug identified in subfolder, and saturating concentrations of fluorescently-labeled GRP-PH domain, a PIP3 product lipid binder, in order to count each product lipid produced. Enzyme rates are calculated as the slope of product lipid created vs. time. 21.5 ± 0.5°C
Images are provided as uncorrected, 16 bit .nd2 files to preserve all original data. To view these .nd2 files:
1. Download ImageJ viewer at (https://imagej.nih.gov/ij/download.html)
2. Download the BioFormats.jar package at (https://www.openmicroscopy.org/bio-formats/downloads/)
3. Open the ImageJ directory after installation and drag the extracted BioFormats.jar package into the "Plugins" subdirectory
4. Open an .nd2 image using imageJ and select opening options when prompted, if desired. If no modifications are desired, select "OK"
5. The 16 bit image will typically look dark and will require brightness adjustment.
6. In the ImageJ toolbar, select Image >> Adjust >> Brightness/Contrast and select "Auto", or manually set the brightness and contrast.