Solid Phase Synthesis of a Functionalized Bis-Peptide Using …
Published 5/15/2012
Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers
1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System
This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.
Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse
Helen and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular, New York University - NYU
Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.
Preparation of Artificial Bilayers for Electrophysiology Experiments
Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University
Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.
Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Department of Physiology and Biophysics, Weill Cornell Medical College
We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.
Method for Measurement of Viral Fusion Kinetics at the Single Particle Level
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 2Howard Hughes Medical Institute, Harvard Medical School
We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.
A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health
The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.
High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method
1UCLA-DOE Institute for Genomics and Proteomics, University of California Los Angeles , 2Department of Physiology, David Geffen School of Medicine, UCLA
Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.
Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties
1Department of Physiology and Biophysics, Weill Cornell Medical College, 2Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University
Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.
Microcontact Printing of Proteins for Cell Biology
Department of Biomedical Engineering, Columbia University
Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.
Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy
Chemistry and Biochemistry, University of California San Diego - UCSD
This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.
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