Biochemistry

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    Protocols in Current Issue
    Proteome Integral Solubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence, and High-Throughput Target Deconvolution

    Chemical proteomics focuses on the drug–target–phenotype relationship for target deconvolution and elucidation of the mechanism of action—key and bottleneck in drug development and repurposing. Majorly due to the limits of using

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    In vitro Assay to Evaluate Cation Transport of Ionophores

    Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion

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    Proteome Integral Solubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence, and High-Throughput Target Deconvolution
    [Abstract]

    Chemical proteomics focuses on the drug–target–phenotype relationship for target deconvolution and elucidation of the mechanism of action—key and bottleneck in drug development and repurposing. Majorly due to the limits of using chemically modified ligands in affinity-based methods, new, unbiased, proteome-wide, and MS-based

    ...
    In vitro Assay to Evaluate Cation Transport of Ionophores
    [Abstract]

    Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we

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    Binding Affinity Measurements Between DNA Aptamers and their Virus Targets Using ELONA and MST
    Authors:  Gregory T. Pawel, Yuan Ma, Yuting Wu, Yi Lu and Ana Sol Peinetti, date: 11/05/2022, view: 486, Q&A: 0
    [Abstract]

    Aptamers have been selected with strong affinity and high selectivity for a wide range of targets, as recently highlighted by the development of aptamer-based sensors that can differentiate infectious from non-infectious viruses, including human adenovirus and SARS-CoV-2. Accurate determination of the binding affinity between the DNA aptamers and

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    A Fluorescence-based Approach Utilizing Self-labeling Enzyme Tags to Determine Protein Orientation in Large Unilamellar Vesicles
    Authors:  Laura Charlotte Paweletz, Sarina Veit and Thomas Günther Pomorski, date: 11/05/2022, view: 461, Q&A: 0
    [Abstract]

    Reconstitution of membrane proteins into large unilamellar vesicles is an essential approach for their functional analysis under chemically defined conditions. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, and its assessment is important prior to functional measurements. Common approaches

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    In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay
    Authors:  Rebecca J. Burge, Katie H. Jameson, Anthony J. Wilkinson and Jeremy C. Mottram, date: 11/05/2022, view: 486, Q&A: 0
    [Abstract]

    Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a

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    X-ray Crystallography: Seeding Technique with Cytochrome P450 Reductase
    Authors:  Bixia Zhang, Jacob A. Lewis, Rishi Hazra and ChulHee Kang, date: 11/05/2022, view: 452, Q&A: 0
    [Abstract]

    Cytochrome P450 reductase (CPR) is a multi-domain protein that acts as a redox partner of cytochrome P450s. The CPR contains a flavin adenine dinucleotide (FAD)–binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting domain. To achieve catalytic events, the FMN-binding domain needs to move relative to the FAD-binding

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    Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli
    Authors:  Phillip Zhu, Ryan A. Mehl and Richard B. Cooley, date: 11/05/2022, view: 548, Q&A: 0
    [Abstract]

    This protocol describes the recombinant expression of proteins in E. coli containing phosphoserine (pSer) installed at positions guided by TAG codons. The E. coli strains that can be used here are engineered with a ∆serB genomic knockout to produce pSer internally at high levels, so no exogenously added pSer is required, and the addition of pSer

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    Single Protein Detection and Imaging with Evanescent Scattering Microscopy
    Authors:  Pengfei Zhang, Lei Zhou, Rui Wang, Xinyu Zhou, Jiapei Jiang, Zijian Wan and Shaopeng Wang, date: 10/20/2022, view: 660, Q&A: 0
    [Abstract]

    Single-molecule measurements provide statistical distributions of molecular properties, in addition to the ensemble averages. Evanescent detection approaches have been widely used for single-molecule detection because the evanescent field can significantly enhance the light-analyte interaction and reduce the background noise. However, current

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    Analysis of the Ubiquitination and Phosphorylation of Vangl Proteins
    Authors:  Di Feng, Ziwei He and Bo Gao, date: 10/20/2022, view: 799, Q&A: 0
    [Abstract]

    The core planar cell polarity (PCP) protein Vang/Vangl, including Vangl1 and Vangl2 in vertebrates, is indispensable during development. Our previous studies showed that the activity of Vangl is tightly controlled by two important posttranslational modifications, ubiquitination and phosphorylation. Vangl is ubiquitinated through an endoplasmic

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    Measuring Intracellular H2O2 in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7
    Authors:  Lianne J. H. C. Jacobs, Michaela N. Hoehne and Jan Riemer, date: 10/20/2022, view: 757, Q&A: 0
    [Abstract]

    Depending on its local concentration, hydrogen peroxide (H2O2) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H2O2 are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of H2O2 in cellular

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