JoVE Chemistry
Origami Inspired Self-assembly of Patterned and Reconfigurable Particles
1Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 2Department of Chemistry, The Johns Hopkins University
We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.
JoVE Bioengineering
Microfluidic Mixers for Studying Protein Folding
1Department of Physics and Astronomy, Michigan State University, 2Department of Mechanical Engineering, Hong Kong University of Science and Technology, 3Center for Biophotonics, University of California, Davis
In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 μs. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).
JoVE Immunology and Infection
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Department of Pharmacology, School of Medicine, New York University
The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.
JoVE General
Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting
1Department of Chemistry, Hunter College, 2Department of Biochemistry, Albert Einstein College of Medicine
This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.
JoVE General
ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells
The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.
JoVE Bioengineering
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.
JoVE General
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.
JoVE General
4D Imaging of Protein Aggregation in Live Cells
Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.
JoVE Immunology and Infection
Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs
1Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, 2Molecular Plant Pathology Laboratory, Agricultural Research Service, United States Department of Agriculture
A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.
JoVE General
Interview: Protein Folding and Studies of Neurodegenerative Diseases
Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
JoVE General
Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.
JoVE General
A Protocol for the Production of KLRG1 Tetramer
Department of Molecular Microbiology and Immunology, Brown University
This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.
JoVE General
The ITS2 Database
1Department of Bioinformatics, Biocenter, University of Würzburg, 2Institute of Pharmacy and Food Chemistry, University of Würzburg
The ITS2 Database is a workbench for phylogenetic inference simultaneously considering sequence and secondary structure of the internal transcribed spacer 2. This includes data collection with accurate annotation, structure prediction, multiple sequence-structure alignment and fast tree calculation. In a nutshell, this workbench simplifies first phylogenetic analyses to a few clicks.
JoVE Clinical and Translational Medicine
How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index
1Department of Psychiatry, University of Geneva School of Medicine, 2Signal Processing Laboratory, École Polytechnique Fédérale de Lausanne, 3Department of Radiology, University Hospital Center and University of Lausanne, 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital
Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.
JoVE General
RNA Secondary Structure Prediction Using High-throughput SHAPE
High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.
JoVE Editorial
January 2012: This Month in JoVE
Here are some highlights from the January 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo
1Department of Biomedical Engineering, Washington University, 2Institute for Information Transmission Problems, Russian Academy of Sciences, 3Department of Mechanical Engineering and Materials Science, Washington University
This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.
JoVE Neuroscience
Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium
A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.
JoVE General
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
School of Biotechnology, Department of Proteomics, Royal Institute of Technology
A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.
JoVE General
Analyzing and Building Nucleic Acid Structures with 3DNA
1Department of Chemistry & Chemical Biology and BioMaPS Institute for Quantitative Biology, Rutgers - The State University of New Jersey, 2Department of Biological Sciences, Columbia University
The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.
JoVE General
Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue
1Department of Structural and Molecular Neuroscience, McLean Hospital, 2Department of Psychiatry, Harvard Medical School, 3Department of Psychiatry, Beth Israel Deaconess Medical Center
We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.
JoVE General
Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington
An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.
JoVE General
Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
1Institute of Chemistry, Technical University of Berlin, 2The Vollum Institute, Oregon Health & Science University
We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.
JoVE Bioengineering
Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules
1Department of Chemistry and Biochemistry, University Of California Santa Barbara, 2Department of Chemistry and Biochemistry, Program in BioMolecular Science and Engineering, University Of California Santa Barbara
"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.
JoVE Bioengineering
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
1Department of Biotechnology, Delft University of Technology, 2Delft Center for Systems and Control, Delft University of Technology
A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.
JoVE General
Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.
1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University, 5Department of Applied Mathematics, Harvard University, 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology
The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.
JoVE Bioengineering
Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets
Molecular Foundry, Lawrence Berkeley National Laboratory
A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.
JoVE General
Intracellular Refolding Assay
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology
In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.
JoVE General
Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators
1Department of Anesthesia, Stanford University School of Medicine, 2Department of Anaesthesiology, University of Mannheim, 3Department of Anaesthesiology, University of Heidelberg
A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.
JoVE General
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina
Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.
JoVE General
The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.
JoVE General
A Protocol for Computer-Based Protein Structure and Function Prediction
1Center for Computational Medicine and Bioinformatics, University of Michigan, 2Center for Bioinformatics and Department of Molecular Bioscience, University of Kansas
Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.
JoVE Bioengineering
Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers
Department of Chemical Engineering, Stanford University
In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.
JoVE General
Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.
JoVE General
Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
Department of Biological Sciences, University of Illinois Chicago - UIC
In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.
JoVE Editorial
February 2013: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here's a look at what's coming up in the February 2013 Issue of Journal of Visualized Experiments (JoVE).
JoVE Neuroscience
Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis
1Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, 2Department of Neurological Surgery, University of California, San Francisco, 3Department of Neurology, University of California, San Francisco
In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.
JoVE General
Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Department of Biochemistry and Cell Biology, State University of New York
Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.
JoVE General
Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41
MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamAΔ41) from M. magneticum AMB-1.
JoVE General
Associated Chromosome Trap for Identifying Long-range DNA Interactions
Medical Service, VA Palo Alto Health Care System , Stanford University School of Medicine
The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.
JoVE General
Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay
A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly –harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.
JoVE Neuroscience
A Caenorhabditis elegans Model System for Amylopathy Study
We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.
JoVE Editorial
The 2008 Lindau Nobel Laureate Meeting: Robert Huber, Chemistry 1988
Robert Huber, 1988 Nobel Laureate in Chemistry, looks to the rising generation of scientists for an “Einstein of biology” to solve the folding problem, one of sciences great mysteries.
JoVE General
Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy
Department of Biology, Howard Hughes Medical Institute, University of Utah
We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.
JoVE General
Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies
We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.
JoVE General
A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries
Microbiology and Immunology, University of British Columbia - UBC
This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.
JoVE General
Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Cell Biology, New York University School of Medicine
We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.
JoVE General
Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography
1School of Biology, Georgia Institute of Technology, 2Department of Molecular Pharmacology, RWTH Aachen University, 3School of Chemistry and Biochemistry, Georgia Institute of Technology
Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 – 90kDa.
JoVE General
Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium
This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.
JoVE Chemistry
Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
1Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne
Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.
More Results...
