Molecular Biology

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    Protocols in Current Issue
    Preparation of Yeast tRNA Sample for NMR Spectroscopy
    Authors:  Marjorie Catala, Alexandre Gato, Carine Tisné and Pierre Barraud, date: 06/20/2020, view: 448, Q&A: 0
    Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In ...
    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: 452, Q&A: 0
    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 ...
    Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
    Authors:  Himasha M. Perera and Michael A. Trakselis, date: 06/20/2020, view: 629, Q&A: 0
    The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication ...
    Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates
    Authors:  Joshua E. Mayfield, Seema Irani and Yan Zhang, date: 06/20/2020, view: 641, Q&A: 0
    Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive ...
    Low-cost and Multiplexable Whole mRNA-Seq Library Preparation Method with Oligo-dT Magnetic Beads for Illumina Sequencing Platforms
    Authors:  Makoto Kashima, Ayumi Deguchi, Ayumi Tezuka and Atsushi J. Nagano, date: 06/20/2020, view: 883, Q&A: 0
    RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we ...
    Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
    Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and ...
    Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
    Authors:  Gloria Muñoz-Fernández, Alberto Jiménez and José Luis Revuelta, date: 06/20/2020, view: 378, Q&A: 0
    The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus ...
    Preparation of Yeast tRNA Sample for NMR Spectroscopy
    Authors:  Marjorie Catala, Alexandre Gato, Carine Tisné and Pierre Barraud, date: 06/20/2020, view: 448, Q&A: 0
    [Abstract] Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological ...
    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: 452, 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 ...
    Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
    Authors:  Himasha M. Perera and Michael A. Trakselis, date: 06/20/2020, view: 629, Q&A: 0
    [Abstract] The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined ...
    Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates
    Authors:  Joshua E. Mayfield, Seema Irani and Yan Zhang, date: 06/20/2020, view: 641, Q&A: 0
    [Abstract] Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques ...
    Low-cost and Multiplexable Whole mRNA-Seq Library Preparation Method with Oligo-dT Magnetic Beads for Illumina Sequencing Platforms
    Authors:  Makoto Kashima, Ayumi Deguchi, Ayumi Tezuka and Atsushi J. Nagano, date: 06/20/2020, view: 883, Q&A: 0
    [Abstract] RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine ...
    Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
    [Abstract] Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to ...
    Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
    Authors:  Gloria Muñoz-Fernández, Alberto Jiménez and José Luis Revuelta, date: 06/20/2020, view: 378, Q&A: 0
    [Abstract] The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In ...
    Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners
    Authors:  Anca F. Savulescu, Stoyan Stoychev, Sipho Mamputha and Musa M. Mhlanga, date: 06/05/2020, view: 799, Q&A: 0
    [Abstract] RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), ...
    Extracellular Vesicles Tracking and Quantification Using CT and Optical Imaging in Rats
    [Abstract] Exosomes, a subtype of extracellular vesicles, are nanovesicles of endocytic origin. Exosomes contain a plethora of proteins, lipids, and genetic materials of parent cells to facilitate intercellular communications. Tracking exosomes in vivo is fundamentally important to understand their biodistribution pattern and the mechanism of ...
    Mouse Adipose Tissue Protein Extraction
    Authors:  Yu A. An and Philipp E. Scherer, date: 06/05/2020, view: 658, Q&A: 0
    [Abstract] As obesity becomes a global epidemic, the metabolism research field is increasingly focusing on studying the physiological and pathological roles of adipose tissues (AT). However, extracting proteins from AT is challenging due to abundant fat content of intracellular lipid droplets. Several commercial kits for extraction of AT proteins are ...



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