Protocols in Current Issue
    Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor
    Authors:  Rubén Martínez-Barricarte, Xiao-Fei Kong and Jean-Laurent Casanova, date: 06/05/2019, view: 158, Q&A: 0
    [Abstract] The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different ...
    Quantification of Cutaneous Ionocytes in Small Aquatic Organisms
    Authors:  Garfield T. Kwan, Shane H. Finnerty, Nicholas C. Wegner and Martin Tresguerres, date: 05/05/2019, view: 342, Q&A: 0
    [Abstract] Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and ...
    Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates
    Authors:  Cristina Colomer-Winter, José A. Lemos and Ana L. Flores-Mireles, date: 03/20/2019, view: 760, Q&A: 0
    [Abstract] Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as ...
    Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes
    Authors:  Katarzyna Potrzebowska, Janne Lehtonen, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 994, Q&A: 0
    [Abstract] To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow ...
    Separation and Visualization of Low Abundant Ubiquitylated Forms
    Authors:  Ramona Schuster, Tânia Simões, Fabian den Brave and Mafalda Escobar-Henriques, date: 11/20/2018, view: 1179, Q&A: 0
    [Abstract] In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the ...
    Enhancement of Mucus Production in Eukaryotic Cells and Quantification of Adherent Mucus by ELISA
    Authors:  Christian Reuter and Tobias A. Oelschlaeger, date: 06/20/2018, view: 2477, Q&A: 1
    [Abstract] The mucosal surfaces of the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye harbor a resident microflora that lives in symbiosis with their host and forms a complex ecosystem. The protection of the vulnerable epithelium is primarily achieved by mucins that form a gel-like structure adherent to the apical ...
    Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings
    Authors:  Agnès Pinel-Galzi, Eugénie Hébrard, Oumar Traoré, Drissa Silué and Laurence Albar, date: 06/05/2018, view: 1850, Q&A: 0
    [Abstract] Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and ...
    Determining Ribosome Translational Status by Ribo-ELISA
    Authors:  Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 2462, Q&A: 0
    [Abstract] The Ribo-ELISA was originally developed to elucidate the basis for the ribopuromycylation method (RPM)-based detection of ribosome bound nascent chains. The Ribo-ELISA enables characterization of the translational status of ribosomes, and can be applied to the discovery of super-ribosomal complexes with novel ribosome associated macromolecules ...
    Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
    Authors:  Katarina Trajkovic, Hyunkyung Jeong and Dimitri Krainc, date: 01/05/2018, view: 2278, Q&A: 0
    [Abstract] Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary ...
    Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
    Authors:  Ana Lechuga, Mónica Berjón-Otero, Margarita Salas and Modesto Redrejo-Rodríguez, date: 01/05/2018, view: 3454, Q&A: 0
    [Abstract] This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules ...

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