Molecular Biology

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    Protocols in Current Issue
    A Yeast Chromatin-enriched Fractions Purification Approach, yChEFs, from Saccharomyces cerevisiae
    [Abstract] We have adapted a previous procedure and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. This methodology allows the easy, reproducible and scalable recovery of proteins associated with chromatin. By using yChEFs, we bypass subcellular fractionation requirements involved when using ...
    SarkoSpin: A Technique for Biochemical Isolation and Characterization of Pathological TDP-43 Aggregates
    Authors:  Manuela Pérez-Berlanga, Florent Laferrière and Magdalini Polymenidou, date: 11/20/2019, view: 934, 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 ...
    Platelet Isolation and Activation Assays
    Authors:  Laura C. Burzynski, Nicholas Pugh and Murray C.H. Clarke, date: 10/20/2019, view: 1166, Q&A: 0
    [Abstract] Platelets regulate hemostasis and are the key determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets circulate in an inactive state, but become activated in response to damage to the endothelium, which exposes thrombogenic material such as collagen to the blood flow. Activation results in a number of responses, ...
    Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells
    Authors:  Xueer Jiang, Ingrid Brust-Mascher and Li-En Jao, date: 08/20/2019, view: 1165, Q&A: 0
    [Abstract] Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of ...
    Purification and Proteomic Analysis of Alphavirus Particles from Sindbis Virus Grown in Mammalian and Insect Cells
    Authors:  Raquel Hernandez, Trevor Glaros, Gabrielle Rizzo and Davis F. Ferreira, date: 05/20/2019, view: 1688, Q&A: 0
    [Abstract] Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to ...
    DNA Immunization Using in vivo Electroporation for Generating Monoclonal Antibodies Against Mouse IL-9R
    [Abstract] Membrane proteins such as cytokine receptors and G protein-coupled receptors can be drug targets. Recently, we have generated specific monoclonal antibodies (mAbs) against the mouse IL-9 receptor (IL-9R) and found that IL-9R on memory B cells have critical roles in T-dependent immune response. So far, most antibodies against cell surface proteins ...
    In vitro Protein-DNA Binding Assay (AlphaScreen® Technology)
    Authors:  Mika Nomoto, Yasuomi Tada and Hironaka Tsukagoshi, date: 02/05/2019, view: 2455, Q&A: 0
    [Abstract] Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide in vivo binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription ...
    Measurement of Redox States of the β3 Integrin Disulfide Bonds by Mass Spectrometry
    Author:  Joyce Chiu, date: 02/05/2019, view: 1636, 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 ...
    Immunohistochemical Staining of TLR4 in Human Skeletal Muscle Samples
    Authors:  Daniela Verzola, Samantha Milanesi, Francesca Costigliolo and Giacomo Garibotto, date: 01/20/2019, view: 1738, Q&A: 0
    [Abstract] Growing evidence suggests the involvement of TLR4, a receptor in the innate immune system, in muscle loss in uremia. Recently, we have evaluated TLR4 in human skeletal muscle from chronic kidney disease patients, by immunohistochemistry and image analysis. Unlike the commonly-used Western blot method, immunohistochemistry allows for the ...
    Production, Titration and Imaging of Zika Virus in Mammalian Cells
    Authors:  Wesley Freppel, Clément Mazeaud and Laurent Chatel-Chaix, date: 12/20/2018, view: 3160, Q&A: 0
    [Abstract] Since the outbreak of Zika virus (ZIKV) in Latin America and the US in 2016, this flavivirus has emerged as a major threat for public health. Indeed, it is now clear that ZIKV is vertically transmitted from the infected mother to the fetus and this may lead to severe neurological development defects including (but not restricted to) neonate ...



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