Molecular Biology

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    Protocols in Current Issue
    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: 156, 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 ...
    Unbiased Screening of Activated Receptor Tyrosine Kinases (RTKs) in Tumor Extracts Using a Mouse Phospho-RTK Array Kit
    Authors:  Julian Naipauer, Lucas E. Cavallin and Enrique A. Mesri, date: 04/20/2019, view: 392, Q&A: 0
    [Abstract] Kaposi’s sarcoma (KS) herpesvirus (KSHV) is a virus that causes KS, an angiogenic AIDS-associated spindle-cell neoplasm, by activating host oncogenic signaling cascades through autocrine and paracrine mechanisms. Many host signaling cascades co-opted by KSHV including PI3K/AKT/mTORC, NFkB and Notch are critical for cell-specific mechanisms of ...
    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: 682, 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 ...
    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: 1092, 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: 755, 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 ...
    In situ, Cell-free Protein Expression on Microarrays and Their Use for the Detection of Immune Responses
    [Abstract] Until recently, whole-proteome microarrays for comprehensive studies of protein interactions were mostly produced by individual cloning and cellular expression of very many open reading frames, followed by protein isolation and purification as well as array production. To overcome this cumbersome process, we have developed a method to generate ...
    Immunohistochemical Staining of TLR4 in Human Skeletal Muscle Samples
    Authors:  Daniela Verzola, Samantha Milanesi, Francesca Costigliolo and Giacomo Garibotto, date: 01/20/2019, view: 822, 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 ...
    Detection of Ligand-binding to Membrane Proteins by Capacitance Measurements
    Authors:  Verena Burtscher, Matej Hotka and Walter Sandtner, date: 01/05/2019, view: 979, Q&A: 0
    [Abstract] In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Accordingly, studying ligand-receptor interactions at the cellular ...
    Production, Titration and Imaging of Zika Virus in Mammalian Cells
    Authors:  Wesley Freppel, Clément Mazeaud and Laurent Chatel-Chaix, date: 12/20/2018, view: 1212, 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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