Biochemistry

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    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 974, Q&A: 0
    [Abstract]

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves multiple DNA binding states, which are usually dependent on nucleotide binding/hydrolysis and are

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    Intracellular IRF5 Dimerization Assay
    Authors:  Cherrie D. Sherman and Betsy J. Barnes, date: 05/20/2021, view: 1927, Q&A: 0
    [Abstract]

    The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5

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    A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins
    Authors:  Javier Aguilera, Salvador Vazquez-Reyes and Jianjun Sun, date: 05/20/2021, view: 2651, Q&A: 0
    [Abstract]

    Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have

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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: 1342, 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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    A Quantitative Assay to Measure Stress Granule Association of Proteins and Peptides in Semi-permeabilized Human Cells
    Authors:  Saskia Hutten and Dorothee Dormann, date: 12/20/2020, view: 1705, Q&A: 0
    [Abstract]

    Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation

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    Native Co-immunoprecipitation Assay to Identify Interacting Partners of Chromatin-associated Proteins in Mammalian Cells
    Authors:  Afzal Husain, Nasim A. Begum, Maki Kobayashi and Tasuku Honjo, date: 12/05/2020, view: 2158, Q&A: 0
    [Abstract]

    Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to

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    Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
    Authors:  Meng-Lun Hsieh, Alice Boulanger, Leslie G. Knipling and Deborah M. Hinton, date: 12/05/2020, view: 951, Q&A: 0
    [Abstract]

    DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional

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    Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP
    Authors:  Doug Tischer and Orion D. Weiner, date: 09/05/2020, view: 2627, Q&A: 0
    [Abstract] Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference ...
    Protocol for Peptide Synthesis on Spectrally Encoded Beads for MRBLE-pep Assays
    Authors:  Jamin B. Hein, Huy Q. Nguyen, Martha Cyert and Polly M. Fordyce, date: 07/05/2020, view: 2284, Q&A: 0
    [Abstract] Every living cell relies on signal transduction pathways comprised of protein-protein interactions (PPIs). In many cases, these PPIs are between a folded protein domain and a short linear motif (SLiM) within an unstructured region of a protein. As a result of this small interaction interface (3-10 amino acids), the affinities of SLiM-mediated ...
    Assessments of HLA-I Specificities of Anti-HLA-I Monoclonal Antibodies Using Solid Phase Bead Arrays
    Authors:  Anita J. Zaitouna, Daniel S. Ramon and Malini Raghavan, date: 06/20/2020, view: 1921, Q&A: 0
    [Abstract] Human leukocyte antigen class I (HLA-I) molecules are a group of structurally-related cell surface proteins with a high degree of variability within the population. While only up to six variants are expressed in an individual person, the whole population contains thousands of different variants. The ability to distinguish specific variants is ...



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