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    Protocols in Current Issue
    Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
    Authors:  Seeyoung Choi and Paul E. Love, date: 01/05/2018, view: 174, Q&A: 0
    Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends ...
    Transplantation of Fecal Microbiota Shaped by Diet
    Authors:  Keiran H. McLeod, Linda Mason and Eliana Mariño, date: 01/05/2018, view: 90, Q&A: 0
    Alterations in diet and gut microbial ecology underlie the pathogenesis of type 1 diabetes (T1D). In the non-obese diabetic (NOD) mouse, we found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces correlated with ...
    High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
    Authors:  Xingmin Aaron Zhang and Thomas F.J. Martin, date: 01/05/2018, view: 153, Q&A: 0
    Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay ...
    Determining Ribosome Translational Status by Ribo-ELISA
    Authors:  Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 258, Q&A: 0
    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 ...
    The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
    Authors:  Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 131, Q&A: 0
    While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ...
    A Rodent Model for Chronic Brain Hypoperfusion Related Diseases: Permanent Bilateral Occlusion of the Common Carotid Arteries (2VO) in Rats
    Authors:  Mei-Ling Yan and Jing Ai, date: 01/05/2018, view: 122, Q&A: 0
    Permanent occlusion of bilateral common carotid arteries (2VO) in rat is considered as a suitable animal model to mimic chronic brain hypoperfusion status, which is proved to be a risk factor to precede the Alzheimer’s disease or/and vascular ...
    Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
    Authors:  Katarina Trajkovic, Hyunkyung Jeong and Dimitri Krainc, date: 01/05/2018, view: 111, Q&A: 0
    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 ...
    Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions
    Authors:  Yohei Katoh, Kentaro Nakamura and Kazuhisa Nakayama, date: 01/05/2018, view: 232, Q&A: 0
    The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation ...
    Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
    Authors:  Seeyoung Choi and Paul E. Love, date: 01/05/2018, view: 174, Q&A: 0
    [Abstract] Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the ...
    Transplantation of Fecal Microbiota Shaped by Diet
    Authors:  Keiran H. McLeod, Linda Mason and Eliana Mariño, date: 01/05/2018, view: 90, Q&A: 0
    [Abstract] Alterations in diet and gut microbial ecology underlie the pathogenesis of type 1 diabetes (T1D). In the non-obese diabetic (NOD) mouse, we found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces correlated with protection from disease. We reconstituted germ free (GF) NOD mice with fecal bacteria from protected ...
    High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
    Authors:  Xingmin Aaron Zhang and Thomas F.J. Martin, date: 01/05/2018, view: 153, Q&A: 0
    [Abstract] Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus ...
    Determining Ribosome Translational Status by Ribo-ELISA
    Authors:  Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 258, 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 ...
    The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
    Authors:  Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 131, Q&A: 0
    [Abstract] While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding ...
    A Rodent Model for Chronic Brain Hypoperfusion Related Diseases: Permanent Bilateral Occlusion of the Common Carotid Arteries (2VO) in Rats
    Authors:  Mei-Ling Yan and Jing Ai, date: 01/05/2018, view: 122, Q&A: 0
    [Abstract] Permanent occlusion of bilateral common carotid arteries (2VO) in rat is considered as a suitable animal model to mimic chronic brain hypoperfusion status, which is proved to be a risk factor to precede the Alzheimer’s disease or/and vascular dementia. In this protocol, we describe how to successfully ligate the bilateral common carotid arteries ...
    Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
    Authors:  Katarina Trajkovic, Hyunkyung Jeong and Dimitri Krainc, date: 01/05/2018, view: 111, 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 ...
    Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions
    Authors:  Yohei Katoh, Kentaro Nakamura and Kazuhisa Nakayama, date: 01/05/2018, view: 232, Q&A: 0
    [Abstract] The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can ...
    Oral Microbiome Characterization in Murine Models
    [Abstract] The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe ...
    A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 12/20/2017, view: 485, Q&A: 0
    [Abstract] In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and ...