Protocols in Current Issue
    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: 104, 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: 780, 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 ...
    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: 1647, 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 ...
    Notch Ligand Binding Assay Using Flow Cytometry
    Authors:  Shweta Varshney and Pamela Stanley, date: 12/05/2017, view: 2299, Q&A: 0
    [Abstract] Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most ...
    Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins
    Authors:  Steffen Lorenz Drees and Mathias Lübben, date: 08/05/2016, view: 4168, Q&A: 0
    [Abstract] Heavy metals can cause damage to biomolecules such as proteins and DNA in multiple ways. Cells therefore strive for keeping intracellular (heavy) metal ions bound to specific proteins that are capable of handling detoxification, export or integration as cofactors. Metal binding proteins usually provide specific coordination sites that bind certain ...
    Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate
    Authors:  Allexa Dow and Sladjana Prisic, date: 03/05/2016, view: 4074, Q&A: 0
    [Abstract] The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant ...
    Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions
    Authors:  Ian Barr and Feng Guo, date: 09/20/2015, view: 12848, Q&A: 2
    [Abstract] Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by ...
    Measurement of Nucleotide Triphosphate Sugar Transferase Activity via Generation of Pyrophosphate
    Authors:  Kaisei Maeda, Rei Narikawa and Masahiko Ikeuchi, date: 04/20/2015, view: 4988, Q&A: 0
    [Abstract] Nucleotide triphosphate (NTP) transferases (EC. 2. 7. 7. X) transfer a nucleoside monophosphate moiety from NTP to another substrate. NTP sugar transferases form a large member of the NTP transferase. There are many variations for the substrate combination of the NTP sugar transferases. It is important to measure the precise enzymatic activity of ...
    A Chemiluminescence Based Receptor-ligand Binding Assay Using Peptide Ligands with an Acridinium Ester Label
    Authors:  Mari Wildhagen, Melinka A. Butenko, Reidunn B. Aalen, Georg Felix and Markus Albert, date: 03/20/2015, view: 5272, Q&A: 0
    [Abstract] Studying the biochemical interaction of ligands with their corresponding receptors requires highly sensitive detection and monitoring of the bound ligand. Classically, radioactively labelled ligands have been widely used as highly sensitive tools for such binding measurements. Disadvantages of radiolabelling include instability of products, high ...
    In vitro Chitin Binding Assay
    Authors:  Geneviève Ball, Frédéric Cadoret and Romé Voulhoux, date: 02/20/2015, view: 5225, Q&A: 0
    [Abstract] Chitin is polymer of N-acetylglucosamine (GlcNAc) found in the exoskeleton of arthropods and the fungal cell wall. GlcNAc is also implicated in bacterial development, adherence, and signal transduction but can also be used as a carbon source. In vitro chitin binding assay is performed to determine the affinity of a purified protein to the ...