Localizing Proteins on Single Trafficking Organelles in 3D with Semisynthetic Gold Labeling and Platinum Replica
Bijeta Prasai1, Justin W Taraska2
1Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Abstract:
The three-dimensional structures of organelles can be visualized at high resolutions using electron microscopy and tomography. Combining genetically encoded tags with tomography enables the specific targeting and detection of identified proteins inside cells. Here, we describe a method for attaching metal-binding gold nanoparticles to proteins genetically tagged with hexa-histidine sequences. We apply this strategy to visualize the position of intracellular proteins on single organelles in unroofed cells with platinum replica electron microscopy at the nanoscale in three dimensions. We have found that this combined method can label and localize proteins with isotropic high precision to generate quantitative maps of protein positions in and around trafficking organelles at the inner plasma membrane of mammalian cells.
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