Protocols in Current Issue
    Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins
    Authors:  Xu Zheng and David Pincus, date: 06/20/2017, view: 672, Q&A: 0
    [Abstract] This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen peptide. Next, the bait protein is recaptured ...
    Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method
    Authors:  Pamela Bielli and Claudio Sette, date: 05/20/2017, view: 893, Q&A: 0
    [Abstract] RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, ...
    Mating Based Split-ubiquitin Assay for Detection of Protein Interactions
    Authors:  Wijitra Horaruang and Ben Zhang, date: 05/05/2017, view: 1162, Q&A: 0
    [Abstract] The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are ...
    Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST)
    Authors:  Ingvild Gudim, Marie Lofstad, Marta Hammerstad and Hans-Petter Hersleth, date: 04/20/2017, view: 1059, Q&A: 0
    [Abstract] This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus ...
    PRODIGY: A Contact-based Predictor of Binding Affinity in Protein-protein Complexes
    Authors:  Anna Vangone and Alexandre M. J. J. Bonvin, date: 02/05/2017, view: 1462, Q&A: 0
    [Abstract] Biomolecular interactions between proteins regulate and control almost every biological process in the cell. Understanding these interactions is therefore a crucial step in the investigation of biological systems and in drug design. Many efforts have been devoted to unravel principles of protein-protein interactions. Recently, we introduced a ...
    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: 1846, 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 ...
    Cell Surface Protein Detection to Assess Receptor Internalization
    Authors:  Magdalena Czarnecka and Joanna Kitlinska, date: 10/20/2016, view: 1509, Q&A: 0
    [Abstract] The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our ...
    Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins
    Authors:  Steffen Lorenz Drees and Mathias Lübben, date: 08/05/2016, view: 2028, 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: 2142, 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 ...
    Identification of RNA-Binding Proteins by RNA Ligand-based cDNA Expression Library Screening
    Authors:  Min Young Kim, Jong Joo Lee and Chul Geun Kim, date: 01/20/2016, view: 1927, Q&A: 0
    [Abstract] We previously reported when a portion of the Requiem (REQ/DPF2) messenger ribonucleic acid (mRNA) 3’ untranslated region (3’UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3’UTR of REQ mRNA plays a physiological role (Kim et al., 2014). To identify trans-acting factors that ...