Molecular Biology

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    Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro
    [Abstract] The stability of the HIV-1 capsid and the spatiotemporal control of its disassembly, a process called uncoating, need to be finely tuned for infection to proceed. Biochemical methods for measuring capsid lattice disassembly in bulk are unable to resolve intermediates in the uncoating reaction. We have developed a single-particle fluorescence ...
    Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion
    [Abstract] In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on ...
    Tandem Affinity Purification of SBP-CBP-tagged Type Three Secretion System Effectors
    Authors:  Laura Berneking, Marie Schnapp, Theresa Nauth and Moritz Hentschke, date: 06/20/2019, view: 620, Q&A: 0
    [Abstract] Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by ...
    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: 1176, 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: 2196, 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: 1760, 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: 3658, 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: 2926, 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 ...



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