JoVE General
Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology
College of Science and Technology, Temple University
The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.
JoVE Bioengineering
Generation and Recovery of β-cell Spheroids From Step-growth PEG-peptide Hydrogels
The following protocol provides techniques for encapsulating pancreatic β-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.
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.
JoVE General
A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation
1Max Planck Institute of Molecular Plant Physiology, 2University of Kaiserslautern
We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.
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
Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels
Department of Physiology and Biophysics, University of California, Irvine (UCI)
This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.
JoVE Bioengineering
Preparation of Complaint Matrices for Quantifying Cellular Contraction
1Institute for Biophysical Dynamics, University of Chicago, 2Physics Department - James Franck Institute, University of Chicago, 3Interdisciplinary Scientist Training Program, University of Chicago
In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.
JoVE Bioengineering
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University
Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.
JoVE General
Pouring and Running a Protein Gel by reusing Commercial Cassettes
1Department of Biology, University of Florida, 2UF Genetics Institute, University of Florida, 3Plant Molecular & Cellular Biology Program, University of Florida
Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.
JoVE General
Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels
Department of Pharmacology, University of Pennsylvania
The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.
JoVE Bioengineering
Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research
1Department of Agricultural and Biological Engineering, Birck-Bindley Physiological Sensing Facility, Purdue University, 2NASA Ames Research Center, 3Department of Chemistry, Pennsylvania State University Hazleton, 4Cooley LLP, 5NASA Life and Physical Sciences, Human Exploration and Operations Mission Directorate, NASA Headquarters
All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.
JoVE General
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
1Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 2Centre for Biological Signalling Studies (bioss) and Biology III, Faculty of Biology, University of Freiburg, 3Department of Molecular Immunology, Max-Planck-Institute of Immunology and Epigenetics
In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.
JoVE General
Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water
Boston Biomedical Research Institute
Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.
JoVE General
Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)
Department of Entomology, Louisiana State University Agricultural Center
We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).
JoVE General
Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia
A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.
JoVE Immunology and Infection
Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System
Department of Immunology, College of Medicine, Mayo Clinic
The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.
JoVE Bioengineering
Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University
Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.
JoVE Bioengineering
On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids
Mechanical Engineering, Stanford University
Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.
JoVE General
Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
1Clinical Research Division, Fred Hutchinson Cancer Research Center - FHCRC, 2Department of Biochemistry and Microbiology, University of Victoria, 3Broad Institute of MIT and Harvard, 4Genome BC Proteomics Centre, University of Victoria, 5Plasma Proteome Institute
Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.
JoVE Neuroscience
Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice
Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, University of Toledo
The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.
JoVE General
Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, 3Department of Neurology, University of California, Los Angeles
Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.
JoVE General
Measuring Peptide Translocation into Large Unilamellar Vesicles
1Department of Chemistry, Wellesley College,
This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.
JoVE Immunology and Infection
Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus
1Department of Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, 2Department of Obstetrics and Gynecology, College of Medicine, University of Arkansas for Medical Sciences, 3Department of Pathology, College of Medicine, University of Arkansas for Medical Sciences
Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.
JoVE Bioengineering
Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery
1Department of Nanomedicine, The Methodist Hospital Research Institute, 2CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology
We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.
JoVE Clinical and Translational Medicine
Quantitative Analysis of Chromatin Proteomes in Disease
1Department of Anesthesiology, David Geffen School of Medicine at UCLA, 2Department of Medicine, David Geffen School of Medicine at UCLA, 3Department of Physiology, David Geffen School of Medicine at UCLA, 4Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah
Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.
JoVE Clinical and Translational Medicine
MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease
1Department of Pharmaceutical Biosciences, Uppsala University, 2Department of Chemical and Biological Engineering, Chalmers University of Technology
Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.
JoVE Clinical and Translational Medicine
Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry
1Sanford-Burnham Medical Research Institute, 2Division of Dermatology, University of California, San Diego, 3VA San Diego Healthcare Center, 4Moores Cancer Center, University of California, San Diego
Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.
JoVE Immunology and Infection
Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells
1Pathology and Laboratory Medicine, University of Wisconsin-Madison, 2Pathobiological Sciences, University of Wisconsin-Madison
Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.
JoVE General
Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences
We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.
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
Cargo Loading onto Kinesin Powered Molecular Shuttles
1Department of Materials Science and Engineering, University of Florida, 2Department of Biomedical Engineering, Columbia University
Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.
JoVE General
Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation
1Department of Chemistry, Tunghai University, 2Department of Biochemistry, University of California, Riverside
MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.
JoVE Neuroscience
Preparation of Oligomeric β-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices
Taub Institute for Research on Alzheimer's Disease and Aging Brain, Columbia University
One feature of Alzheimer's Disease is the elevation of Aβ1-42 peptide. Here we provide a protocol for preparing synthetic Aβ1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of Aβ-induced pathology and drug screening.
JoVE General
Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
1Department of Biochemistry / Analytics, caprotec bioanalytics GmbH, 2Institute of Organic Chemistry, RWTH Aachen University
Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.
JoVE General
Fabrication and Use of MicroEnvironment microArrays (MEArrays)
1Life Science Division, Lawrence Berkeley National Laboratory, 2Department of Comparative Biochemistry, University of California, Berkeley
A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.
JoVE Immunology and Infection
A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors
1Department of Entomology, Texas A&M University (TAMU), 2Department of Molecular and Cellular Medicine, Texas A&M University (TAMU)
This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.
JoVE General
PuraMatrix Encapsulation of Cancer Cells
1Wellman Center for Photomedicine Massachusetts General Hospital, Harvard Medical School, 2Thayer School of Engineering, Dartmouth College, 3Department of Dermatology, Harvard Medical School
This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel.
JoVE Neuroscience
Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants
1Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, 2Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, 3Division of Genetics, Department of Medicine, Boston Children's Hospital, 4Howard Hughes Medical Institute, Boston Children's Hospital, 5Department of Pathology, Boston Children's Hospital, Harvard Medical School
The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.
JoVE Bioengineering
Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering
1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 2Biomedical Sciences Program, University of California, San Diego, 3Department of Bioengineering, University of California, San Diego
Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues
JoVE General
Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid
Protein Expression and Purification Core Facility, EMBL Heidelberg
A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.
JoVE Immunology and Infection
A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms
1Department of Biology, University of Texas San Antonio - UTSA, 2South Texas Center for Emerging Infectious Diseases, University of Texas San Antonio - UTSA
We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.
JoVE General
Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes
1Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biomedical Engineering and Cardiac Bioelectricity and Arrhythmia Center, Washington University in St. Louis
Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.
JoVE Bioengineering
High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Chemistry, Iowa State University
In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.
JoVE General
Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
1Department of Cell Biology, Emory University, 2Department of Medicine, Division of Cardiology, Emory University
The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.
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
A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples
1Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco - UCSF, 2Buck Institute for Age Research, 3Department of Chemistry, Purdue University
Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.
JoVE Neuroscience
Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS
Department of Surgery, University of Toronto
Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.
JoVE General
Measuring Fast Calcium Fluxes in Cardiomyocytes
1Department of Biological Sciences and Geology, Queensborough Community College, 2Department of Physiology & Biophysics, Stony Brook University
We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.
JoVE Clinical and Translational Medicine
Skeletal Muscle Gender Dimorphism from Proteomics
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College
A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.
JoVE General
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto
Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.
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