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Semi-quantitative Determination of Protein Expression Using Immunohistochemistry Staining and Analysis: An Integrated Protocol

Authors: Alexandra R Crowe and Wei Yue, date: 12/20/2019, view: 662, Q&A: 0

[Abstract] Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed ...

Separation of Natural Collagen Crosslinks Using Buffer and Ion-pairing Agent Free Solvents on Silica Hydride Column for Mass Spectrometry Detection

Authors: Rafea Naffa and Joseph Pesek, date: 05/05/2019, view: 992, Q&A: 0

[Abstract] In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation ...

Measurement of Redox States of the β3 Integrin Disulfide Bonds by Mass Spectrometry

Author: Joyce Chiu, date: 02/05/2019, view: 1516, Q&A: 0

[Abstract] Functional disulfide bonds mediate a change in protein function in which they reside when cleaved or formed. To elucidate how a functional disulfide bond controls protein activity, it is critical that the redox state of the bond in the population of protein molecules is known. Measurement of changes in disulfide bond redox state relies on thiol ...

Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages

Authors: Gaurav Kumar, Subhash B. Arya and Amit Tuli, date: 12/20/2018, view: 2019, Q&A: 0

[Abstract] Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs ...

Relative Quantitation of Polymerized Actin in Suspension Cells by Flow Cytometry

Authors: Mallory R. Kakley, Katrina B. Velle and Lillian K. Fritz-Laylin, date: 11/20/2018, view: 2132, Q&A: 0

[Abstract] The amount of polymerized actin within a cell can vary widely due to natural processes and/or experimentally induced perturbations. We routinely use this protocol to measure relative polymerized actin content between cell populations by staining cells in suspension with fluorescent phalloidin, then measuring total cell fluorescence using flow ...

Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans

Authors: Alejandro Mata-Cabana, Olga Sin, Renée I. Seinstra and Ellen A. A. Nollen, date: 10/20/2018, view: 3129, Q&A: 0

[Abstract] C. elegans is widely used to investigate biological processes related to health and disease. To study protein localization, fluorescently-tagged proteins can be used in vivo or immunohistochemistry can be performed in whole worms. Here, we describe a technique to localize a protein of interest at a subcellular level in C. elegans ...

Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates

Authors: Olga Sin, Alejandro Mata-Cabana, Renée I. Seinstra and Ellen A. A. Nollen, date: 10/05/2018, view: 3179, Q&A: 0

[Abstract] Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein’s native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a C. elegans model of polyglutamine diseases, we ...

A Procedure for Precise Determination of Glutathione Produced by Saccharomyces cerevisiae

Authors: Jyumpei Kobayashi, Daisuke Sasaki and Akihiko Kondo, date: 06/20/2018, view: 3264, Q&A: 0

[Abstract] In bioproduction, yields of products must be calculated precisely for accurate evaluation of various fermentation conditions. To evaluate productivity of microorganisms, product amounts per unit of medium volume (e.g., mg-product/L-broth), and/or product amounts per unit of a microorganism amount (e.g., mg-product/mg-dry cell ...

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: 3090, 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 ...

Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue

Authors: Alicia A Cutler, Anita H Corbett and Grace K Pavlath, date: 12/20/2017, view: 5912, Q&A: 0

[Abstract] Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. ...

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