Biochemistry

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    Protocols in Current Issue
    Supported Cell Membrane Sheets to Monitor Protein Assembly
    Authors:  Simon Erlendsson, Thor Seneca Thorsen and Kenneth Lindegaard Madsen, date: 09/20/2019, view: 40, Q&A: 0
    [Abstract] Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, ...
    Cell-based Assay for Recruitment of DDR1 to Collagen-coated Beads
    Authors:  Victoria Juskaite and Birgit Leitinger, date: 08/20/2019, view: 284, Q&A: 0
    [Abstract] The discoidin domain receptors, DDR1 and DDR2, are key signaling receptors for the extracellular matrix protein collagen. The interactions of cells with collagen are difficult to study because of the difficulty to obtain native collagen fibers for in vitro studies. Thus, in vitro studies often use acid-soluble collagens in the ...
    Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide
    Authors:  Anthony Yammine, Jinlong Gao and Ann H. Kwan, date: 06/05/2019, view: 1492, Q&A: 0
    [Abstract] Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. ...
    Cross-linking, Immunoprecipitation and Proteomic Analysis to Identify Interacting Proteins in Cultured Cells
    Authors:  Hao Wang, Meiling He, Belinda Willard and Qingyu Wu, date: 06/05/2019, view: 1030, Q&A: 0
    [Abstract] Extracellular expression is essential for the function of secreted and cell surface proteins. Proper intracellular trafficking depends on protein interactions in multiple subcellular compartments. Co-immunoprecipitation and the yeast two-hybrid system are commonly used to investigate protein-protein interactions. These methods, however, depend on ...
    An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein
    Authors:  Yili Zhu, Weimin Tan and Wei-Lih Lee, date: 12/05/2018, view: 1728, Q&A: 0
    [Abstract] In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by ...
    Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels
    Authors:  Philipp A.M. Schmidpeter and Crina M. Nimigean, date: 10/05/2018, view: 2122, Q&A: 0
    [Abstract] The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by ...
    Isothermal Titration Calorimetry: A Biophysical Method to Characterize the Interaction between Label-free Biomolecules in Solution
    Author:  Andrea Saponaro, date: 08/05/2018, view: 8185, Q&A: 0
    [Abstract] This protocol can be applied to analyze the direct interaction between a soluble protein and a target ligand molecule using Isothermal Titration Calorimetry (ITC, Malvern). ITC allows the biophysical characterization of binding between label-free, non-immobilized and in-solution biomolecules by providing the stoichiometry of the interaction, the ...
    Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
    Authors:  Yuichi Ichikawa and Paul D. Kaufman, date: 03/20/2018, view: 2935, Q&A: 0
    [Abstract] Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the ...
    Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins
    Authors:  Luke Berry, Angela Patterson, Natasha Pence, John W. Peters and Brian Bothner, date: 03/20/2018, view: 3775, Q&A: 0
    [Abstract] The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen ...
    An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
    [Abstract] Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these ...



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