Molecular Biology

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    Protocols in Current Issue
    Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates
    Authors:  Cristina Colomer-Winter, José A. Lemos and Ana L. Flores-Mireles, date: 03/20/2019, view: 683, Q&A: 0
    [Abstract] Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as ...
    In situ, Cell-free Protein Expression on Microarrays and Their Use for the Detection of Immune Responses
    [Abstract] Until recently, whole-proteome microarrays for comprehensive studies of protein interactions were mostly produced by individual cloning and cellular expression of very many open reading frames, followed by protein isolation and purification as well as array production. To overcome this cumbersome process, we have developed a method to generate ...
    Cluster Analysis of Endogenous HER2 and HER3 Receptors in SKBR3 Cells
    [Abstract] The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with ...
    Identifying Protein Interactions with Histone Peptides Using Bio-layer Interferometry
    Authors:  Bingbing Ren, Ahmed Mahmoud Mohammed Sayed, Hwei Ling Tan, Yu Keung Mok and Ee Sin Chen, date: 09/20/2018, view: 1597, Q&A: 0
    [Abstract] Histone post-translational modifications (PTMs) regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair. Most histone PTMs affect the recruitment or exclusion of reader proteins from chromatin. Here, we present a protocol to measure affinity and interaction kinetics between histone peptides and the ...
    Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation
    Authors:  Tyler Milston Renner, Kasandra Bélanger and Marc-André Langlois, date: 09/05/2018, view: 1234, Q&A: 0
    [Abstract] There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly ...
    α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
    Authors:  Michael M. Wördehoff and Wolfgang Hoyer, date: 07/20/2018, view: 2790, Q&A: 1
    [Abstract] Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (e.g., aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive ...
    Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE
    Authors:  Marjaana Rantala, Virpi Paakkarinen and Eva-Mari Aro, date: 07/05/2018, view: 2205, Q&A: 0
    [Abstract] The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis ...
    CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii
    Authors:  Shaojun Long, Kevin M. Brown and L. David Sibley, date: 03/20/2018, view: 3923, Q&A: 0
    [Abstract] Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this ...
    Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer
    Authors:  Nils Y. Meiresonne, Svetlana Alexeeva, René van der Ploeg and Tanneke den Blaauwen, date: 01/20/2018, view: 3490, Q&A: 0
    [Abstract] This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Förster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent ...
    MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
    Authors:  Maximilian G. Plach, Klaus Grasser and Thomas Schubert, date: 12/05/2017, view: 8184, Q&A: 0
    [Abstract] Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides ...



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