Protocols in Current Issue
    Visualization and Purification of Caenorhabditis elegans Germ Granule Proteins Using Proximity Labeling
    Authors:  Hannah L. Hertz, Ian F. Price and Wen Tang, date: 04/20/2022, view: 1404, Q&A: 0

    Membraneless organelles, such as germ granules and stress granules, are liquid-like condensates formed by phase transition. Recently, we and others have adopted proximity-based labeling methods to determine the composition of these membraneless compartments. Here, we describe the use of TurboID—an engineered promiscuous biotin

    Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy
    Authors:  Kevin Singewald, James A. Wilkinson and Sunil Saxena, date: 12/20/2021, view: 1442, Q&A: 0

    Site-directed spin labeling in conjunction with electron paramagnetic resonance (EPR) is an attractive approach to measure residue specific dynamics and point-to-point distance distributions in a biomolecule. Here, we focus on the labeling of proteins with a Cu(II)-nitrilotriacetic acid (NTA) complex, by exploiting two strategically placed

    Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE
    Authors:  Joel Weinberger II and Christopher W. Lennon, date: 08/20/2021, view: 2327, Q&A: 0

    Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by

    A New Method for Studying RNA-binding Proteins on Specific RNAs
    Authors:  Weiping Sun, Ziheng Zhang, Ji-Long Liu and Min Zhuang, date: 05/20/2021, view: 8110, Q&A: 0

    Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT.

    In vitro Induction and Detection of Acrosomal Exocytosis in Human Spermatozoa
    Authors:  Shenae L. Cafe, Amanda L. Anderson and Brett Nixon, date: 07/20/2020, view: 2652, Q&A: 0
    [Abstract] The acrosome reaction is a highly regulated exocytotic event that primes spermatozoa for successful fertilization. Upon induction, acrosomal exocytosis proceeds via a wave of vesiculation that radiates across the sperm head, destabilizing the acrosomal vesicle and resulting in the release of the acrosomal contents. Having shed their acrosome, ...
    Imaging VIPER-labeled Cellular Proteins by Correlative Light and Electron Microscopy
    [Abstract] Advances in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for studying diverse proteins and nanostructures involved in fundamental cell biology. It is now possible to visualize and quantify the spatial organization of cellular proteins and other macromolecules by FM, ...
    Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy
    Authors:  Julia K. Doh, Caroline A. Enns and Kimberly E. Beatty, date: 11/05/2019, view: 3854, Q&A: 0
    [Abstract] Genetically-encoded tags are useful tools for multicolor and multi-scale cellular imaging. Versatile Interacting Peptide (VIP) tags, such as VIPER, are new genetically-encoded tags that can be used in various imaging applications. VIP tags consist of a coiled-coil heterodimer, with one peptide serving as the genetic tag and the other (“probe ...
    Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins
    Authors:  Julia K. Doh, Savannah J. Tobin and Kimberly E. Beatty, date: 11/05/2019, view: 3739, Q&A: 0
    [Abstract] Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag (Doh et al., 2018), which contains two elements: a genetically-encoded peptide tag (i.e., CoilE) and a probe peptide (i.e., ...
    Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides
    Authors:  Masatoshi Murai and Hideto Miyoshi, date: 09/05/2019, view: 3292, Q&A: 0
    [Abstract] The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [125 ...
    Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila
    [Abstract] Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as ‘click chemistry’ serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small ...

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