Protocols in Current Issue
    Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins

    Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires’ disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids

    Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
    Authors:  Meng-Lun Hsieh, Alice Boulanger, Leslie G. Knipling and Deborah M. Hinton, date: 12/05/2020, view: 708, Q&A: 0

    DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional

    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: 4727, 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 ...
    Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
    Authors:  Yuichi Ichikawa and Paul D. Kaufman, date: 03/20/2018, view: 4762, 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 ...
    Producing GST-Cbx7 Fusion Proteins from Escherichia coli
    Authors:  Thao Ngoc Huynh and Xiaojun Ren , date: 06/20/2017, view: 7729, Q&A: 1
    [Abstract] This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside ...
    Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry
    Authors:  Manish Bhuwan, Madhuri Suragani, Nasreen Z. Ehtesham and Seyed E. Hasnain, date: 01/20/2017, view: 8998, Q&A: 0
    [Abstract] The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway ...
    Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins
    Authors:  Steffen Lorenz Drees and Mathias Lübben, date: 08/05/2016, view: 8129, 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: 7385, 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 ...
    Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae
    Authors:  Gang Li, Margarita Marroquin-Guzman and Richard A. Wilson, date: 11/05/2015, view: 11675, Q&A: 0
    [Abstract] Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an ...
    Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores
    Authors:  Jidnyasa Ingale and Richard T Wyatt, date: 10/05/2015, view: 6563, Q&A: 0
    [Abstract] Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer ...

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