Biochemistry

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    Protocols in Current Issue
    Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies
    Authors:  Si Liu and Jiangtao Guo, date: 04/05/2021, view: 146, Q&A: 0

    Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+

    ...
    In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
    Authors:  Yanjun Li, Di Sun, Xingxing Yan, Zhiye Wang and Xiuren Zhang, date: 04/05/2021, view: 174, Q&A: 0

    The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the

    ...
    Imaging Microtubules in vitro at High Resolution while Preserving their Structure
    Authors:  Camille Cuveillier, Yasmina Saoudi, Isabelle Arnal and Christian Delphin, date: 04/05/2021, view: 159, Q&A: 0

    Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule

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    Atomic Force Microscopy to Characterize Ginger Lipid-Derived Nanoparticles (GLDNP)
    Authors:  Dingpei Long, Chunhua Yang, Junsik Sung and Didier Merlin, date: 04/05/2021, view: 148, Q&A: 0

    We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive

    ...
    FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution

    Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of

    ...
    Reconstitution of Chromatin by Stepwise Salt Dialysis
    Authors:  Grisel Cruz-Becerra and James T. Kadonaga, date: 04/05/2021, view: 127, Q&A: 0

    Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields

    ...
    Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies
    Authors:  Si Liu and Jiangtao Guo, date: 04/05/2021, view: 146, Q&A: 0
    [Abstract]

    Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney.

    ...
    In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
    Authors:  Yanjun Li, Di Sun, Xingxing Yan, Zhiye Wang and Xiuren Zhang, date: 04/05/2021, view: 174, Q&A: 0
    [Abstract]

    The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be

    ...
    Imaging Microtubules in vitro at High Resolution while Preserving their Structure
    Authors:  Camille Cuveillier, Yasmina Saoudi, Isabelle Arnal and Christian Delphin, date: 04/05/2021, view: 159, Q&A: 0
    [Abstract]

    Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection

    ...
    Atomic Force Microscopy to Characterize Ginger Lipid-Derived Nanoparticles (GLDNP)
    Authors:  Dingpei Long, Chunhua Yang, Junsik Sung and Didier Merlin, date: 04/05/2021, view: 148, Q&A: 0
    [Abstract]

    We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive components, including lipids, proteins, miRNAs, and ginger secondary metabolites (gingerols and shogaols).

    ...
    FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution
    [Abstract]

    Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we

    ...
    Reconstitution of Chromatin by Stepwise Salt Dialysis
    Authors:  Grisel Cruz-Becerra and James T. Kadonaga, date: 04/05/2021, view: 127, Q&A: 0
    [Abstract]

    Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for

    ...
    Monitoring Real-time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism
    Authors:  Martin Balcerowicz, Marco Di Antonio and Betty Y. W. Chung, date: 03/20/2021, view: 955, Q&A: 0
    [Abstract]

    RNA secondary structures are highly dynamic and subject to prompt changes in response to the environment. Temperature in particular has a strong impact on RNA structural conformation, and temperature-sensitive RNA hairpin structures have been exploited by multiple organisms to modify the rate of translation in response to temperature changes.

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    Quantitative Measurement of Mucolytic Enzymes in Fecal Samples
    Authors:  Shahanshah Khan and Hasan Zaki, date: 03/20/2021, view: 679, Q&A: 0
    [Abstract]

    The mucus layer in the gastrointestinal tract covers the apical surface of intestinal epithelial cells, protecting the mucosal tissue from enteric pathogen and commensal microorganisms. The mucus is primarily composed of glycosylated protein called mucins, which are produced by goblet cells, a type of columnar epithelial cells in the intestinal

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    Simultaneous Imaging of Single Protein Size, Charge, and Binding Using A Protein Oscillation Approach
    Authors:  Guangzhong Ma, Zijian Wan and Shaopeng Wang, date: 03/05/2021, view: 1204, Q&A: 0
    [Abstract]

    Electrophoresis and Western blot are important tools in protein research for detection and identification of proteins. These traditional techniques separate the proteins based on size and charge differences and identify the proteins by antibody binding. Over the past decade, the emergence of single-molecule techniques has shown great potential in

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    Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE)
    Authors:  Wolf-Matthias Leeder, Elisabeth Kruse and H. Ulrich Göringer, date: 03/05/2021, view: 399, Q&A: 0
    [Abstract]

    Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is

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