Molecular Biology

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    Protocols in Current Issue
    Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells
    Authors:  Jingen Zhu, Pan Tao, Marthandan Mahalingam and Venigalla B. Rao, date: 04/05/2020, view: 84, Q&A: 0
    A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The ...
    Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila
    Authors:  Océane Marescal, Frieder Schöck and Nicanor González-Morales, date: 04/05/2020, view: 90, Q&A: 0
    Protein-protein interactions in Drosophila myofibrils are essential for their function and formation. Bimolecular Fluorescence Complementation (BiFC) is an effective method for studying protein interactions and localization. BiFC relies on ...
    Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages
    Authors:  Moumita Basu, Shriya Saha and Anindita Ukil, date: 04/05/2020, view: 93, Q&A: 0
    Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. ...
    Real-time Fluorescence Measurement of Enterovirus Uncoating
    Authors:  Visa Ruokolainen, Mira Laajala and Varpu Marjomäki, date: 04/05/2020, view: 75, Q&A: 0
    Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of ...
    Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
    Authors:  Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 115, Q&A: 0
    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 ...
    Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins
    Authors:  Tyler B. Johnson, Jiri Adamec and Paul Blum, date: 04/05/2020, view: 113, Q&A: 0
    High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine ...
    Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale
    Authors:  Paul O. Sheridan, Jennifer C. Martin and Karen P. Scott, date: 04/05/2020, view: 58, Q&A: 0
    Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of ...
    Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay
    Authors:  Connor M. Blair, Jiayue Ling and George S. Baillie, date: 04/05/2020, view: 44, Q&A: 0
    Cyclic nucleotide degrading phosphodiesterase (PDE) enzymes are crucial to the fine tuning of cAMP signaling responses, playing a pivotal role in regulating the temporal and spatial characteristics of discrete cAMP nanodomains and hence the activity ...
    Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells
    Authors:  Jingen Zhu, Pan Tao, Marthandan Mahalingam and Venigalla B. Rao, date: 04/05/2020, view: 84, Q&A: 0
    [Abstract] A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we ...
    Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila
    Authors:  Océane Marescal, Frieder Schöck and Nicanor González-Morales, date: 04/05/2020, view: 90, Q&A: 0
    [Abstract] Protein-protein interactions in Drosophila myofibrils are essential for their function and formation. Bimolecular Fluorescence Complementation (BiFC) is an effective method for studying protein interactions and localization. BiFC relies on the reconstitution of a monomeric fluorescent protein from two half-fragments when in proximity. Two ...
    Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages
    Authors:  Moumita Basu, Shriya Saha and Anindita Ukil, date: 04/05/2020, view: 93, Q&A: 0
    [Abstract] Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different ...
    Real-time Fluorescence Measurement of Enterovirus Uncoating
    Authors:  Visa Ruokolainen, Mira Laajala and Varpu Marjomäki, date: 04/05/2020, view: 75, Q&A: 0
    [Abstract] Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative ...
    Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
    Authors:  Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 115, 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 ...
    Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins
    Authors:  Tyler B. Johnson, Jiri Adamec and Paul Blum, date: 04/05/2020, view: 113, Q&A: 0
    [Abstract] High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol ...
    Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale
    Authors:  Paul O. Sheridan, Jennifer C. Martin and Karen P. Scott, date: 04/05/2020, view: 58, Q&A: 0
    [Abstract] Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic ...
    Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay
    Authors:  Connor M. Blair, Jiayue Ling and George S. Baillie, date: 04/05/2020, view: 44, Q&A: 0
    [Abstract] Cyclic nucleotide degrading phosphodiesterase (PDE) enzymes are crucial to the fine tuning of cAMP signaling responses, playing a pivotal role in regulating the temporal and spatial characteristics of discrete cAMP nanodomains and hence the activity of cAMP-effector proteins. As a consequence of orchestrating cAMP homeostasis, dysfunctional PDE ...
    Single-cell qPCR Assay with Massively Parallel Microfluidic System
    Authors:  Marta Prieto-Vila, Takahiro Ochiya and Yusuke Yamamoto, date: 03/20/2020, view: 229, Q&A: 0
    [Abstract] The single-cell transcriptome is the set of messenger RNA molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Due to sensitivity shortages, most of genetic studies use bulk samples, providing only the average gene expression. Single-cell technologies have provided a ...
    High-resolution and Deep Phylogenetic Reconstruction of Ancestral States from Large Transcriptomic Data Sets
    Authors:  Sumanth Kumar Mutte and Dolf Weijers, date: 03/20/2020, view: 319, Q&A: 0
    [Abstract] Phylogenetics is an important area of evolutionary biology that helps to understand the origin and divergence of genes, genomes and species. Building meaningful phylogenetic trees is needed for the accurate reconstruction of the past. To achieve a correct phylogenetic understanding of genes or proteins, reliable and robust methods are needed to ...



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