Biochemistry

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    Protocols in Current Issue
    Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP
    Authors:  Anwar Sadat, Satyam Tiwari and Koyeli Mapa, date: 07/20/2021, view: 56, Q&A: 0
    [Abstract]

    Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the

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    Monitoring Changes in the Oxidizing Milieu in the Endoplasmic Reticulum of Mammalian Cells Using HyPerER
    Authors:  Julia Birk, Beata Lizak, Christian Appenzeller-Herzog and Alex Odermatt, date: 07/05/2021, view: 1206, Q&A: 0
    [Abstract]

    The production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress are tightly linked. The generation of ROS can be both the cause and a consequence of ER stress pathways, and an increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. For the assessment of modulators

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    Immunofluorescence of GFAP and TNF-α in the Mouse Hypothalamus
    Authors:  Prasad S. Dalvi and Denise D. Belsham, date: 07/05/2021, view: 596, Q&A: 0
    [Abstract]

    Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome

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    Intracellular IRF5 Dimerization Assay
    Authors:  Cherrie D. Sherman and Betsy J. Barnes, date: 05/20/2021, view: 1880, Q&A: 0
    [Abstract]

    The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5

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    Imaging Microtubules in vitro at High Resolution while Preserving their Structure
    Authors:  Camille Cuveillier, Yasmina Saoudi, Isabelle Arnal and Christian Delphin, date: 04/05/2021, view: 718, Q&A: 0
    [Abstract]

    Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection

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    Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells
    Authors:  Shaista Anwer and Katalin Szaszi, date: 07/20/2020, view: 1484, Q&A: 0
    [Abstract] Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows ...
    Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures
    Authors:  Johanna M. Schafer and Jennifer A. Pietenpol, date: 07/05/2020, view: 1614, Q&A: 0
    [Abstract] MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system ...
    Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations
    Authors:  Terra M. Kuhn, Shawn C. Little and Maya Capelson, date: 07/05/2020, view: 5199, Q&A: 0
    [Abstract] Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal ...
    Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
    Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 1457, Q&A: 0
    [Abstract] Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and ...
    FRET Reporter Assays for cAMP and Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in Living Cells
    Authors:  Brandon T. Milliken, Robert P. Doyle, George G. Holz and Oleg G. Chepurny, date: 06/05/2020, view: 2641, Q&A: 0
    [Abstract] Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca2+. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors ...



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