Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by
1Laboratory of Fungal Pathogenesis, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500039, Telangana, India.
Abstract:
Phosphatidylinositol 3-phosphate (PI3P) levels govern membrane trafficking in Candida glabrata. Here, we present a confocal imaging-based protocol for PI3P localization analysis using the GFP-FYVE (found in Fab1, YOTB, Vac1, and EEA1) fusion protein. We describe steps for cloning the FYVE domain into the GFP-containing vector backbone, transforming FYVE-GFP into C. glabrata, and preparing slides with FYVE-GFP-expressing C. glabrata cells. We then detail procedures for acquiring and analyzing images and quantifying signal data. This protocol is adaptable to subcellular localization analysis of other low-abundant lipid and protein molecules. For complete details on the use and execution of this protocol, please refer to Askari et al. (2023).1.
More Related Videos
07:26Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes
Published on: October 15, 2016
08:07Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
Published on: July 26, 2019