Biochemistry

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    Protocols in Current Issue
    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

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    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

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    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: 175, 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

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    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.

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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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    Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP)
    Authors:  Marisa Müller, Tamas Schauer and Peter B. Becker, date: 03/05/2021, view: 920, Q&A: 0
    [Abstract]

    RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target

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    Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells
    Authors:  Natalia Pacheco-Fernandez, Mehrshad Pakdel and Julia von Blume, date: 03/05/2021, view: 1040, Q&A: 0
    [Abstract]

    Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling,

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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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    Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
    Authors:  Ryota Futamata, Noriyuki Kioka and Kazumitsu Ueda, date: 02/20/2021, view: 1029, Q&A: 0
    [Abstract]

    The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of

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    Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
    Authors:  Wenjie Zhao, Xudong Wang and Libin Ye, date: 02/20/2021, view: 1353, Q&A: 0
    [Abstract]

    In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains,

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