Proteolysis:
Cleavage of proteins into smaller peptides or amino acids either by Proteases or non-enzymatically (e.g., Hydrolysis). It does not include Protein Processing, Post-Translational.
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 Clinical and Translational Medicine
MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression
1Department of Pharmacology, Wayne State University, 2Barbara Ann Karmanos Cancer Institute, Wayne State University
We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.
JoVE General
Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg
Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.
JoVE General
Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Faculty of Life Sciences, University of Manchester
Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.
JoVE General
Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis
1Whitehead Institute for Biomedical Research, 2Department of Biology, MIT - Massachusetts Institute of Technology, 3Howard Hughes Medical Institute
SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.
JoVE Editorial
August 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens-free "on-chip" microscopy platform, which they have pioneered.
JoVE General
Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation
This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.
JoVE Editorial
The 2009 Lindau Nobel Laureate Meeting: Aaron Ciechanover, Chemistry 2004
Aaron Ciechanover was born in Haifa, Israel in October 1947. Ciechanover shared the Nobel Prize in Chemistry in 2004 with Avram Hershko and Irwin Rose for their discovery of ubiquitin-mediated protein degradation.
JoVE General
Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts
We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.
JoVE Clinical and Translational Medicine
Modeling and Imaging 3-Dimensional Collective Cell Invasion
1Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 2The Beatson Institute for Cancer Research
Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.
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
Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
1Molecular and Cell Biology, Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center
A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.
JoVE General
Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos
Institute for Cell and Molecular Biosciences, University of Newcastle, United Kingdom
The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.
JoVE Immunology and Infection
Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, 3Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), 4Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System
An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.
JoVE Bioengineering
A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System
1Institute for BioNanotechnology in Advanced Medicine, Northwestern University, 2Department of Obstetrics and Gynecology, Northwestern University, Feinberg School of Medicine, 3Center for Reproductive Research, Northwestern University, 4The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 5Department of Chemical and Biological Engineering, Northwestern University
A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.
JoVE General
Western Blotting: Sample Preparation to Detection
Research and Development, EMD Chemicals Inc.
Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.
JoVE Clinical and Translational Medicine
Acute Myocardial Infarction in Rats
1Department of Internal Medicine, Division of Cardiology, University of Texas Medical Branch, 2Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston (UH), Texas Medical Center
The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.
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 Clinical and Translational Medicine
Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain
1Department of Surgery, University of Texas Medical Branch, 2Department of Neuroscience and Cell Biology, University of Texas Medical Branch
A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.
JoVE General
Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
Molecular, Cellular and Developmental Biology, University of Michigan
A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.
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
Cellular Encapsulation in 3D Hydrogels for Tissue Engineering
1Department of Bioengineering, University of Pennsylvania, 2Department of Bioengineering, University of Pennsylvania-School of Medicine
We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.
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 Bioengineering
Viral Nanoparticles for In vivo Tumor Imaging
1Department of Biomedical Engineering, Case Western Reserve University, 2Department of Biomedical Engineering, Radiology, and Materials Science and Engineering, Case Western Reserve University
Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.
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.
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 Clinical and Translational Medicine
Spheroid Assay to Measure TGF-β-induced Invasion
An assay to quantitatively measure Transforming Growth Factor (TGF)-β-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.
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 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 Neuroscience
Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient
1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities, University of Wisconsin
A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.
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 Editorial
November 2011: This Month in JoVE
Here are some highlights from the November 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
Purification of Hsp104, a Protein Disaggregase
Department of Biochemistry and Biophysics, University of Pennsylvania
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
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