Biochemistry

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    Protocols in Current Issue
    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: 101, 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: 113, 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., ...
    F-actin Bundle Sedimentation Assay
    Authors:  Shan-Shan Lin, Mei-Chun Chuang and Ya-Wen Liu, date: 11/05/2019, view: 99, Q&A: 0
    [Abstract] Understanding the molecular mechanism governing the higher-order regulation of actin dynamics requires chemically-defined and quantitative assays. Recently, the membrane remodeling large GTPase, dynamin, has been identified as a new actin cross-linking molecule. Dynamin regulates actin cytoskeleton through binding to, self-assembling around, and ...
    Isolation and Imaging of His- and RFP-tagged Amyloid-like Proteins from Caenorhabditis elegans by TEM and SIM
    Authors:  Amberley D. Stephens, Meng Lu and Gabriele S. Kaminski Schierle, date: 11/05/2019, view: 179, Q&A: 0
    [Abstract] In our recently published paper, we highlight that during normal aging of C. elegans age-dependent aggregates of proteins form and lead to functional decline of tissues. The protocol described here details the isolation of two proteins from C. elegans in their aggregated amyloid-like form, casein kinase I isoform alpha (KIN-19) ...
    Measuring Small-molecule Inhibition of Protein Interactions in Live Cells Using FLIM-FRET
    Authors:  James M. Pemberton, Qian Liu and David W. Andrews, date: 10/20/2019, view: 1126, Q&A: 0
    [Abstract] This protocol was designed to quantitatively measure small-molecule displacement of proteins in live mammalian cells using fluorescence lifetime imaging microscopy–Förster resonance energy transfer (FLIM-FRET). Tumour cell survival is often dependent on anti-apoptotic proteins, which bind to and inhibit pro-apoptotic proteins, thus preventing ...
    Immunohistochemical Identification of Muscle Fiber Types in Mice Tibialis Anterior Sections
    Authors:  Vanitha V. Rao and Abhishek Mohanty, date: 10/20/2019, view: 260, Q&A: 0
    [Abstract] Mammalian skeletal muscle is a metabolically active tissue that is made up of different types of muscle fibers. These myofibers are made up of important contractile proteins that provide force during contraction of the muscle like actin and myosin. Murine myofibers have been classified into 4 types: Type I, Type IIa, Type IIb and Type IIX. Each ...
    A Highly Sensitive, Reproducible Assay for Determining 4-hydroxynonenal Protein Adducts in Biological Material
    Authors:  T. Blake Monroe and Ethan J. Anderson, date: 10/05/2019, view: 284, Q&A: 0
    [Abstract] Oxidative stress is associated with numerous diseases, and markers of oxidative stress in biological material are becoming a mainstay of both experimental and clinical/epidemiological research. Lipid peroxidation is a major form of oxidative stress, but due to their rapid degradation and instability, lipid peroxides are notoriously difficult to ...
    Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites
    Authors:  Amalia Papadaki and Haralabia Boleti, date: 10/05/2019, view: 231, Q&A: 0
    [Abstract] Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through ...
    Conjugation of Fab’ Fragments with Fluorescent Dyes for Single-molecule Tracking on Live Cells
    Authors:  I-Ting Teng, Xiangning Bu and Inhee Chung, date: 09/20/2019, view: 382, Q&A: 0
    [Abstract] Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have ...



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