Electrophoresis:
An electrochemical process in which macromolecules or colloidal particles with a net electric charge migrate in a solution under the influence of an electric current.
JoVE General
Agarose Gel Electrophoresis for the Separation of DNA Fragments
Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles
A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.
JoVE Neuroscience
Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay
1Department of Radiation Oncology, University of Alabama-Birmingham, 2Department of Radiation Oncology, The Ohio State University Medical School, 3Department of Cell Biology, and Pharmacology and Toxicology, Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, University of Alabama-Birmingham
The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.
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
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
A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles
Department of Biochemistry and Molecular Biology, University of British Columbia
Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.
JoVE General
Optimized PCR-based Detection of Mycoplasma
Product Management, Sigma-Aldrich
The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.
JoVE General
Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes
Research and Development, GE Healthcare Bio-Sciences AB
A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan™ DIGE technology.
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 General
Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells
David Geffen School of Medicine, University of California, Los Angeles
In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.
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 Application Notes
Simultaneous, Rapid, and Highly Efficient Protein Transfer Using the Trans-Blot Turbo Transfer System - ADVERTISEMENT
The Trans-Blot Turbo system reduces protein transfer protocols from gels to as little as 3 minutes, while maintaining high efficiency transfers and high throughput. The system enables protein transfer of 2 mini gels in 3 minutes and up to 4 mini gels in as little as 7 minutes.
JoVE General
Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels
Biological Medical Research Center (BMFZ), University Duesseldorf
This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.
JoVE General
Streamlined Purification of Plasmid DNA From Prokaryotic Cultures
This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.
JoVE General
Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR
The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University
An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.
JoVE General
DNA-based Fish Species Identification Protocol
This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.
JoVE General
Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)
1School of Biological Sciences, Nanyang Technological University, Singapore - NTU, 2Singapore-MIT Alliance for Reserach and Technology (SMART)
Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.
JoVE General
Electrophoretic Separation of Proteins
Proteomic Center, Keck Graduate Institute of Applied Life Sciences
In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).
JoVE General
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
1Department of Biological Sciences, University of Alabama Huntsville, 2Department of Biology, Stanford University
A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.
JoVE General
A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica
1Department of Biological Sciences, The University of Alabama, Huntsville, 2USDA-APHIS-PPQ, Center for Plant Health Science and Technology
We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).
JoVE Immunology and Infection
DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
Department of Microbiology, Immunology and Pathology, Colorado State University
Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.
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 Neuroscience
Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease
1Department of Neurology, David Geffen School of Medicine, 2Molecular Biology Institute, University of California, Los Angeles, 3Brain Research Institute, University of California, Los Angeles
Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
JoVE Immunology and Infection
An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
Department of Population Health, University of Georgia
We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.
JoVE General
Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences
Department of Cell and Developmental Biology, University of Pennsylvania
We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.
JoVE Immunology and Infection
Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens
1Department of Laboratory Medicine, University of California, San Francisco, 2Division of Infectious Diseases, University of California, San Francisco
The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.
JoVE General
Extraction of High Molecular Weight DNA from Microbial Mats
We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.
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
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
Genotyping of Plant and Animal Samples without Prior DNA Purification
Thermo Scientific Molecular Biology Products, Thermo Fisher Scientific
The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.
JoVE Immunology and Infection
Detection of Bacteria Using Fluorogenic DNAzymes
1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Department of Chemistry and Chemical Biology, McMaster University
We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).
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
Microfluidic Chips Controlled with Elastomeric Microvalve Arrays
Dept. of Bioengineering, University of Washington
We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.
JoVE General
Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana
1Department of Energy - Plant Research Laboratory, Michigan State University (MSU), 2Department of Biochemistry and Molecular Biology, Michigan State University (MSU)
The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.
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 Immunology and Infection
Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry
Department of Chemistry, Stony Brook University
Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.
JoVE General
DNA Methylation: Bisulphite Modification and Analysis
1Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, 2St Vincent's Clinical School, University of NSW
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
JoVE Bioengineering
Microfluidic Chip Fabrication and Method to Detect Influenza
1Department of Mechanical Engineering, Boston University, 2Department of Biomedical Engineering, Boston University
An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.
JoVE General
Electroeluting DNA Fragments
This procedure allows the purification of DNA fragments with high yield.
JoVE Immunology and Infection
Examination of the Telomere G-overhang Structure in Trypanosoma brucei
Biological, Geo. & Env. Sciences, Cleveland State University
Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.
JoVE General
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Centre for Infectious Diseases and Microbiology, University of Sydney
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
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
Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells
Gene Expression Division, Bio-Rad Laboratories
This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.
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
Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules
1Department of Physics, Texas A&M University (TAMU), 2Department of Biomedical Engineering, Texas A&M University (TAMU)
We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.
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 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 Clinical and Translational Medicine
Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract
1St. Erik's Eye Hospital, Karolinska Institutet, 2Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University
Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.
JoVE General
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City
This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.
JoVE Bioengineering
Graphene Coatings for Biomedical Implants
1Department of Physics, Clemson University, 2Department of Pharmacology and Toxicology, East Carolina University, 3Department of Bioengineering, Clemson University, 4Center for Optical Materials Science and Engineering Technologies, Clemson University
Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.
JoVE Applied Physics
Compact Quantum Dots for Single-molecule Imaging
1Department of Biomedical Engineering, Emory University, 2Department of Chemistry, Georgia Institute of Technology
We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.
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