Biochemistry

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    Protocols in Current Issue
    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: 823, 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: 628, 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 ...
    Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners
    Authors:  Anca F. Savulescu, Stoyan Stoychev, Sipho Mamputha and Musa M. Mhlanga, date: 06/05/2020, view: 962, Q&A: 0
    [Abstract] RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), ...
    In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes
    Authors:  Hanako Nishikawa, Masaru Sasaki and Ken-ichi Nishiyama, date: 05/20/2020, view: 1074, Q&A: 0
    [Abstract] It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in E. coli using purified factors, in which the ...
    Measurement of Protein-Protein Interactions through Microscale Thermophoresis (MST)
    Authors:  Magnez Romain, Bryan Thiroux, Morgane Tardy, Bruno Quesnel and Xavier Thuru, date: 04/05/2020, view: 1115, Q&A: 0
    [Abstract] The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations resulted in controversy regarding the values of the dissociation constant (Kd) (Freeman et al., 2000). MST is a powerful new method for the ...
    Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria
    Authors:  Bo Ma, Chao Fang, Jing Zhang, Mingzhi Wang, Xiaoxing Luo and Zheng Hou, date: 03/05/2020, view: 957, Q&A: 0
    [Abstract] The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to ...
    Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation
    Authors:  Claudia Cattoglio, Iryna Pustova, Xavier Darzacq, Robert Tjian and Anders S. Hansen, date: 02/20/2020, view: 1557, Q&A: 0
    [Abstract] Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins ...
    Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol
    Authors:  Bodan Hu, Mohamed Rasheed Gadalla, Christoph Thiele and Michael Veit, date: 02/20/2020, view: 1244, Q&A: 0
    [Abstract] Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a cholesterol derivative in living cells or with immunoprecipitated protein. We synthesized a “clickable” ...
    Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1
    Authors:  Wai Wa Ray Chan, Dik Long Dennis Chau, Wen Li and Kwok-Fai Lau, date: 12/05/2019, view: 1339, Q&A: 0
    [Abstract] Intramolecular interaction is a common mechanism that regulates protein activities. Conventionally, such interactions are investigated by classical in vitro biochemical assays. Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation ...
    Detection of in vivo Protein Interactions in All Bacterial Compartments by Förster Resonance Energy Transfer with the Superfolder mTurquoise2 ox-mNeongreen FRET Pair
    Authors:  Nils Y. Meiresonne, Elisa Consoli, Laureen M.Y. Mertens and Tanneke den Blaauwen, date: 12/05/2019, view: 1574, Q&A: 0
    [Abstract] This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in all compartments of Escherichia coli by Förster Resonance Energy Transfer (FRET) using the Superfolder mTurquoise2 ox-mNeonGreen FRET pair (sfTq2ox-mNG). This FRET pair has more than twice the detection range for FRET ...



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