Molecular Biology


    Protocols in Current Issue
    A High-throughput Automated ELISA Assay for Detection of IgG Antibodies to the SARS-CoV-2 Spike Protein

    The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein. The assay robustly distinguishes positive from negative

    Optimised Method for the Production and Titration of Lentiviral Vectors Pseudotyped with the SARS-CoV-2 Spike

    The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike

    Differential Analysis of N-glycopeptide Abundance and N-glycosylation Site Occupancy for Studying Protein N-glycosylation Dysregulation in Human Disease
    Authors:  Qi Zhang, Cheng Ma, Lian Li and Lih-Shen Chin, date: 06/20/2021, view: 1885, Q&A: 0

    Protein N-glycosylation plays a vital role in diverse cellular processes, and dysregulated N-glycosylation is implicated in a variety of human diseases including neurodegenerative disorders and cancer. With recent advances in high-resolution mass spectrometry-based glycoproteomics technologies enabling large-scale N-glycoproteome profiling of

    In vitro Measurement of Membrane Attack Complex in RPE Cells
    Authors:  Kelly Mulfaul and Sarah L. Doyle, date: 02/20/2021, view: 2464, Q&A: 0

    Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the

    Assembly and Imaging Set up of PIE-Scope
    [Abstract] Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy ...
    Quantification of the Surface Expression of G Protein-coupled Receptors Using Intact Live-cell Radioligand Binding Assays
    Authors:  Xin Xu and Guangyu Wu, date: 09/20/2020, view: 2880, Q&A: 0
    [Abstract] G protein-coupled receptors (GPCRs) are the most structurally diverse family of signaling proteins and regulate a variety of cell function. For most GPCRs, the cell surface is their functional destination where they are able to respond a wide range of extracellular stimuli, leading to the activation of intracellular signal transduction cascades. ...
    A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
    Authors:  Natarajan V. Bhanu, Simone Sidoli and Benjamin A Garcia, date: 09/20/2020, view: 3418, Q&A: 0
    [Abstract] Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have ...
    Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres
    Authors:  Szilárd Szikora, Tibor Novák, Tamás Gajdos, Miklós Erdélyi and József Mihály, date: 06/20/2020, view: 3141, Q&A: 0
    [Abstract] Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful ...
    Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease
    Authors:  Yibing Deng, Shengchao Shi, Bin Zheng, Tao Wu and Peng Zheng, date: 04/20/2020, view: 3172, Q&A: 0
    [Abstract] The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the ...
    Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
    Authors:  Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 2803, Q&A: 0
    [Abstract] This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (Em = -55 mV) in the SdhA flavoprotein subunit of succinate ...

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