Titlebook: Membrane Trafficking; Methods and Protocol Jingshi Shen Book 2022 The Editor(s) (if applicable) and The Author(s), under exclusive license

MEEK

Generation of Endogenously Tagged Membrane Trafficking Regulators Using CRISPR Genome Editingosed proteins. Biochemical and cell biological studies of vesicle trafficking often require the introduction of epitope tags or fluorescent protein markers for protein purification and tracking in cells. Previously, such tagging experiments in mammalian cells mainly used overexpression systems, whic

BULLY

Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting vesicles to the plasma membrane. In the assay, we use a plasmid that encodes a fusion protein of the mitochondria protein Tom20 and Sec3 N-terminally tagged with the florescence protein mCherry, and coexpress the plasmid in yeast cells with CIT1-GFP, a marker protein of mitochondria. We then detect

Amplify

GLARE

Live-Cell Superresolution Imaging of Retrograde Axonal Trafficking Using Pulse–Chase Labeling in Culafficking. Further, hippocampal neurons exhibit “en passant” presynapses that may confound the analysis of long-range retrograde axonal transport. To solve these issues, we and others have developed microfluid-based methods to specifically follow the fates of the retrograde axonal cargoes following

新鲜

Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapseynaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and bio

财产

In Vitro Reconstitution Studies of SNAREs and Their Regulators Mediating GLUT4 Vesicle Fusionoteins. For example, synip and tomosyn negatively regulate GLUT4 SNARE-mediated membrane fusion. Here we describe in vitro reconstituted assays to determine the molecular mechanisms of SNAREs, synip, and tomosyn. These methods can also be extended to the studies of other types of membrane fusion eve

染色体

Imaging Single-Vesicle Exocytosis with Total Internal Reflection Fluorescence Microscopy (TIRFM)ws for visualization of membrane dynamics at the cell surface with superb spatiotemporal resolution. In this chapter, TIRFM is used to record and analyze exocytosis of single glucose transporter-4 (GLUT4) containing vesicles in 3T3-L1 adipocytes.

污秽

Localizing Proteins on Single Trafficking Organelles in 3D with Semisynthetic Gold Labeling and Platncoded 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 in

Hemiplegia

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