Molecular Biology

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    Protocols in Current Issue
    Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro
    [Abstract] The stability of the HIV-1 capsid and the spatiotemporal control of its disassembly, a process called uncoating, need to be finely tuned for infection to proceed. Biochemical methods for measuring capsid lattice disassembly in bulk are unable to resolve intermediates in the uncoating reaction. We have developed a single-particle fluorescence ...
    In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria
    Authors:  Krishna K. Singh, Devendra P. Singh, Rambir Singh and Deepak K. Saini, date: 07/05/2019, view: 205, Q&A: 0
    [Abstract] Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic ...
    Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion
    [Abstract] In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on ...
    Preparation and Purification of Active Recombinant Human Pancreatic Lipase in Escherichia coli
    Authors:  Nanami Kawaguchi, Haruko Ogawa and Kimie Date, date: 07/05/2019, view: 194, Q&A: 0
    [Abstract] Human pancreatic lipase (HPL) is the main lipolytic enzyme involved in the digestion of dietary fat. An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time in an Escherichia coli (E. coli) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from ...
    Imaging Gene Expression Dynamics in Pseudomonas fluorescens In5 during Interactions with the Fungus Fusarium graminearum PH-1
    Authors:  Rosanna C. Hennessy, Peter Stougaard and Stefan Olsson, date: 06/20/2019, view: 372, Q&A: 0
    [Abstract] Genomics, transcriptomics and metabolomics are powerful technologies for studying microbial interactions. The main drawback of these methods is the requirement for destructive sampling. We have established an alternative but complementary technique based on a microplate system combined with promoter fusions for visualizing gene expression in space ...
    Production and Purification of Dengue Virus-like Particles from COS-1 Cells
    Authors:  Jedhan Ucat Galula, Gwong-Jen J. Chang and Day-Yu Chao, date: 06/20/2019, view: 340, Q&A: 0
    [Abstract] Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. ...
    Tandem Affinity Purification of SBP-CBP-tagged Type Three Secretion System Effectors
    Authors:  Laura Berneking, Marie Schnapp, Theresa Nauth and Moritz Hentschke, date: 06/20/2019, view: 344, Q&A: 0
    [Abstract] Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by ...
    Transient Expression Assay in Strawberry Fruits
    Authors:  Mengting Pi, Qi Gao and Chunying Kang, date: 06/05/2019, view: 492, Q&A: 0
    [Abstract] Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, ...
    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: 455, 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: 580, 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 ...



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