Protocols in Current Issue
    Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
    Authors:  Francesca Mattiroli, Yajie Gu and Karolin Luger, date: 02/05/2018, view: 151, Q&A: 0
    [Abstract] Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack ...
    Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus
    Authors:  Anthony J. Snyder and Pranav Danthi, date: 01/20/2018, view: 179, Q&A: 0
    [Abstract] Mammalian orthoreovirus (reovirus) utilizes pore forming peptides to penetrate host cell membranes. This step is essential for delivering its genome containing core particle during viral entry. This protocol describes an in vitro assay for measuring reovirus-induced pore formation.
    Infectious Subviral Particle to Membrane Penetration Active Particle (ISVP-to-ISVP*) Conversion Assay for Mammalian Orthoreovirus
    Authors:  Anthony J. Snyder and Pranav Danthi, date: 01/20/2018, view: 180, Q&A: 0
    [Abstract] The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of conformational changes prior to or during viral entry. These transitions are necessary for delivering the genome-containing core across host cell membranes. This protocol describes an in vitro assay for monitoring the transition into a membrane penetration-active ...
    Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
    Authors:  Seeyoung Choi and Paul E. Love, date: 01/05/2018, view: 340, Q&A: 0
    [Abstract] Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the ...
    Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER
    Authors:  Simon Matthé Erstad and Yumiko Sakuragi, date: 01/05/2018, view: 307, Q&A: 0
    [Abstract] Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding ...
    Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
    Authors:  Nathanael P. Cottam and Daniel Ungar, date: 11/20/2017, view: 743, Q&A: 0
    [Abstract] Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was ...
    γ-Secretase Epsilon-cleavage Assay
    [Abstract] γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO ...
    In vitro NLK Kinase Assay
    Authors:  Sungho Moon, Jiyoung Kim and Eek‐hoon Jho, date: 11/05/2017, view: 724, Q&A: 0
    [Abstract] This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.
    Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity
    [Abstract] Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by ...
    In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Authors:  Grant Schauer, Jeff Finkelstein and Mike O’Donnell, date: 09/20/2017, view: 1372, Q&A: 0
    [Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other ...