To determine the subcellular localization of a protein of interest, the protein can be fused to a fluorescent protein, e.g. GFP. The localization can be analyzed by confocal microscopy.
Method Article
Confocal microscopy
https://doi.org/10.1038/nprot.2007.127
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To determine the subcellular localization of a protein of interest, the protein can be fused to a fluorescent protein, e.g. GFP. The localization can be analyzed by confocal microscopy.
For our equipment, an incident Argon (488 nm) ion laser (Coherent Inc.) beam was coupled to a modified Yokogawa spinning disc confocal scan head (Yokogawa Electric, Japan; and Solamere Technology, USA) via an acoustical optical tunable filter (NEOS, USA). The confocal head was mounted on an inverted microscope (DM IRE2, Leica, Germany) equipped with a 63x water immersion objective (n.a.-1.3 HCX PL APO 21°C, Leica, Germany) and a motorized Z-stage. Fluorescence images (525/50-nm for GFP) were acquired (500 msec acquisition time) with a cooled on-chip multiplication gain Cascade 512B digital camera (Roper Scientific, Tucson, AZ). Instrumentation was driven using Metamorph Version 6.3r3 software (Molecular Devices, Sunnyvale, CA).
Grow yeast cells transformed with GFP-fusion on solid media.
Transfer cells to a microscope slide and cover with a coverslip.
Acquire Z-sections using a custom-made spinning disk confocal microscope as described above.
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