Molecular Biology

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    Protocols in Current Issue
    Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics
    Authors:  Lite Yang, Ivan Tochitsky, Clifford J. Woolf and William Renthal, date: 08/05/2021, view: 24, Q&A: 0

    Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of

    ...
    CRISPR-mediated Labeling of Cells in Chick Embryos Based on Selectively Expressed Genes
    Authors:  Masahito Yamagata and Joshua R. Sanes, date: 08/05/2021, view: 62, Q&A: 0

    The abilities to mark and manipulate specific cell types are essential for an increasing number of functional, structural, molecular, and developmental analyses in model organisms. In a few species, this can be accomplished by germline transgenesis;

    ...
    Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC

    Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered;

    ...
    Purification and Cryo-electron Microscopy Analysis of Plant Mitochondrial Ribosomes
    Authors:  Florent Waltz, Philippe Giegé and Yaser Hashem, date: 08/05/2021, view: 18, Q&A: 0

    Plants make up by far the largest part of biomass on Earth. They are the primary source of food and the basis of most drugs used for medicinal purposes. Similarly to all eukaryotes, plant cells also use mitochondria for energy production. Among

    ...
    Fe-NTA Microcolumn Purification of Phosphopeptides from Immunoprecipitation (IP) Eluates for Mass Spectrometry Analysis
    Authors:  Ethan J. Sanford and Marcus B. Smolka, date: 08/05/2021, view: 14, Q&A: 0

    Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often

    ...
    A Fast and Easy Method to Study Ralstonia solanacearum Virulence upon Transient Gene Expression or Gene Silencing in Nicotiana benthamiana Leaves
    Authors:  Wenjia Yu and Alberto P. Macho, date: 08/05/2021, view: 20, Q&A: 0

    Ralstonia solanacearum is a devastating soil-borne bacterial pathogen that causes disease in multiple host plants worldwide. Typical assays to measure virulence of R. solanacearum in laboratory conditions rely on soil-drenching inoculation followed

    ...
    A Fast and Easy Method to Study Ralstonia solanacearum Virulence upon Transient Gene Expression or Gene Silencing in Nicotiana benthamiana Leaves
    Authors:  Wenjia Yu and Alberto P. Macho, date: 08/05/2021, view: 20, Q&A: 0
    [Abstract]

    Ralstonia solanacearum is a devastating soil-borne bacterial pathogen that causes disease in multiple host plants worldwide. Typical assays to measure virulence of R. solanacearum in laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a novel inoculation protocol to

    ...
    Fe-NTA Microcolumn Purification of Phosphopeptides from Immunoprecipitation (IP) Eluates for Mass Spectrometry Analysis
    Authors:  Ethan J. Sanford and Marcus B. Smolka, date: 08/05/2021, view: 14, Q&A: 0
    [Abstract]

    Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often missing phosphorylation events from low-abundance proteins. In order to increase coverage of

    ...
    Purification and Cryo-electron Microscopy Analysis of Plant Mitochondrial Ribosomes
    Authors:  Florent Waltz, Philippe Giegé and Yaser Hashem, date: 08/05/2021, view: 18, Q&A: 0
    [Abstract]

    Plants make up by far the largest part of biomass on Earth. They are the primary source of food and the basis of most drugs used for medicinal purposes. Similarly to all eukaryotes, plant cells also use mitochondria for energy production. Among mitochondrial gene expression processes, translation is the least understood; although, recent advances

    ...
    Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC
    [Abstract]

    Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long

    ...
    CRISPR-mediated Labeling of Cells in Chick Embryos Based on Selectively Expressed Genes
    Authors:  Masahito Yamagata and Joshua R. Sanes, date: 08/05/2021, view: 62, Q&A: 0
    [Abstract]

    The abilities to mark and manipulate specific cell types are essential for an increasing number of functional, structural, molecular, and developmental analyses in model organisms. In a few species, this can be accomplished by germline transgenesis; in other species, other methods are needed to selectively label somatic cells based on the genes

    ...
    Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics
    Authors:  Lite Yang, Ivan Tochitsky, Clifford J. Woolf and William Renthal, date: 08/05/2021, view: 24, Q&A: 0
    [Abstract]

    Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of peripheral sensory neurons has been limited by both their heterogeneity and low abundance compared with

    ...
    GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish
    [Abstract]

    Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging)

    ...
    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 1129, Q&A: 0
    [Abstract]

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA

    ...
    A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells
    Authors:  Amanda L. Koch, Tatsuya Morisaki and Timothy J. Stasevich, date: 07/20/2021, view: 859, Q&A: 0
    [Abstract]

    Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind

    ...
    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 1060, Q&A: 0
    [Abstract]

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves multiple DNA binding states, which are usually dependent on nucleotide binding/hydrolysis and are

    ...



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