Solid Phase Synthesis of a Functionalized Bis-Peptide Using …
Published 5/15/2012
Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy
Department of Molecular and Cellular Biology, University of California Davis
We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.
Electron Cryotomography of Bacterial Cells
1Division of Biology, California Institute of Technology - Caltech, 2Howard Hughes Medical Institute, California Institute of Technology - Caltech
We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.
Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques
1Department of Immunology, MD Anderson Cancer Center - University of Texas, 2Department of Medicine, University of Miami
We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.
Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique
1Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 2Carolina Center for Nanotechnology Excellence, University of North Carolina
This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.
Crystallization of Membrane Proteins in Lipidic Mesophases
Molecular Biology, The Scripps Research Institute
The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.
In vivo Electroporation of Developing Mouse Retina
1Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, 2Department of Neurology, Johns Hopkins School of Medicine, 3Department of Ophthalmology, Johns Hopkins School of Medicine, 4Center for High-Throughput Biology, Johns Hopkins School of Medicine, 5Institute for Cell Engineering, Johns Hopkins School of Medicine
A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.
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.
Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation
Department of Pathology and Immunology, Washington University School of Medicine
This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.
Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge , 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia , 7Duke University , 8Yale University, 9University of Notre Dame , 10Washington University in St. Louis , 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology
The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.
Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Department of Structural Biology, University of Pittsburgh School of Medicine
This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.
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