Biochemistry

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    Protocols in Current Issue
    SarkoSpin: A Technique for Biochemical Isolation and Characterization of Pathological TDP-43 Aggregates
    Authors:  Pérez-Berlanga Manuela, Laferrière Florent and Polymenidou Magdalini, date: 11/20/2019, view: 131, Q&A: 0
    TDP-43 is the main aggregating protein in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Aggregated TDP-43 is resistant to diverse detergent solubilization, yet physiological ...
    ELISA Based Protein Ubiquitylation Measurement
    Authors:  Yuka Kamada, Ryosuke Fukuda and Tsukasa Okiyoneda, date: 11/20/2019, view: 17, Q&A: 0
    Ubiquitylation is a common post-translational modification of cellular proteins that results in proteasomal and lysosomal degradations. Ubiquitylation is generally measured by methods such as immunoblotting using anti-ubiquitin antibodies after ...
    SarkoSpin: A Technique for Biochemical Isolation and Characterization of Pathological TDP-43 Aggregates
    Authors:  Pérez-Berlanga Manuela, Laferrière Florent and Polymenidou Magdalini, date: 11/20/2019, view: 131, Q&A: 0
    [Abstract] TDP-43 is the main aggregating protein in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Aggregated TDP-43 is resistant to diverse detergent solubilization, yet physiological TDP-43 and other abundant proteins commonly co-purify with pathological TDP-43. This mixed isolation has ...
    ELISA Based Protein Ubiquitylation Measurement
    Authors:  Yuka Kamada, Ryosuke Fukuda and Tsukasa Okiyoneda, date: 11/20/2019, view: 17, Q&A: 0
    [Abstract] Ubiquitylation is a common post-translational modification of cellular proteins that results in proteasomal and lysosomal degradations. Ubiquitylation is generally measured by methods such as immunoblotting using anti-ubiquitin antibodies after isolating the protein-of-interest by denaturing immunoprecipitation. The following protocol can be used ...
    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: 128, 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: 147, 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: 124, 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: 232, 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) ...
    Amplex Red Assay for Measuring Hydrogen Peroxide Production from Caenorhabditis elegans
    Authors:  Ozgur Karakuzu, Melissa R. Cruz, Yi Liu and Danielle A. Garsin, date: 11/05/2019, view: 139, Q&A: 0
    [Abstract] Reagents such as Amplex® Red have been developed for detecting hydrogen peroxide (H2O2) and are used to measure the release of H2O2 from biological samples such as mammalian leukocytes undergoing the oxidative burst. Caenorhabditis elegans is commonly used as a model host in the study ...
    Purification and HPLC Analysis of Cell Wall Muropeptides from Caulobacter crescentus
    Authors:  Gabriele Stankeviciute and Eric A. Klein, date: 11/05/2019, view: 149, Q&A: 0
    [Abstract] The peptidoglycan sacculus, or cell wall, is what defines bacterial cell shape. Cell wall composition can be best characterized at the molecular level by digesting the peptidoglycan murein polymer into its muropeptide subunits and quantifying the abundance of muropeptides using high-pressure liquid chromatography. Certain features of the cell wall ...
    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: 1171, 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 ...



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