Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.
Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response
1Department of Biological Sciences, University of Alabama
Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.
1INSERM UMR 1043, CNRS UMR 5282, Université Toulouse 3, 2UMR Viroligie, INRA ENVA ANSES
These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.
1Department of Engineering and Neuroscience Program, Trinity College
Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely behaving mouse models of neuropathology.
Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters
1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Department of Molecular Biology, Princeton University, 3Division of Chemistry and Chemical Engineering, California Institute of Technology
This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.
1Institute for Research on Cancer and Ageing of Nice, UMR CNRS 7284 - INSERM, U1081 - UNS, Centre Antoine Lacassagne, University of Nice - Sophia Antipolis
We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.
1Center for Molecular Bacteriology and Infection, Imperial College London
The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.
1Department of Medicine, University of Dresden, 2Center for Regerative Therapies Dresden
Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
1Department of Genetics, Albert Einstein College of Medicine, 2Shanghai Key Laboratory of Complex Prescription and MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine
Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.
Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose
1Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology
The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.