Buckles, Thomas (2022). CIL: 52028, Homo sapiens. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J0DR2V0V

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.

• 2022

• Buckles, Thomas

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.

Imaging mode: Fluoresence
Visualization methods: objective based TIRFM using a modified Nikon TE-2000U (60x 1.49 N.A. TIRF objective) and illumination from a 532-nm solid-state laser with power modification by neutral-density filters

• Cell Image Library Group ID: 20034

• Homo sapiens

• Cell polarity establishment and maintenance

• data
• image

• No linguistic content; Not applicable

Samplenumber: 52028

Source data

• Source Record in the Cell Image Library: https://doi.org/10.7295/W9CIL52028

Creative Commons Attribution 4.0 International Public License

• UC Regents

Under copyright (US)

Use: This work is available from the UC San Diego Library. This digital copy of the work is intended to support research, teaching, and private study.

Constraint(s) on Use: This work is protected by the U.S. Copyright Law (Title 17, U.S.C.). Use of this work beyond that allowed by "fair use" or any license applied to this work requires written permission of the copyright holder(s). Responsibility for obtaining permissions and any use and distribution of this work rests exclusively with the user and not the UC San Diego Library. Inquiries can be made to the UC San Diego Library program having custody of the work.

Research Data Curation Program, UC San Diego, La Jolla, 92093-0175 (https://lib.ucsd.edu/rdcp)

2022-11-18