1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University
An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.
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.
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.
1Department of Physical Electronics, Faculty of Engineering, Tel Aviv University, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biological Sciences, University of Illinois, 4Department of Cell Research and Immunology, Tel Aviv University
Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.
This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.
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.
Bio-Plex® overview with TGF-β mention
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.
Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.
Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells
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.
Gene Expression Division, Bio-Rad Laboratories
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.
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.
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.
Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
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.
We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.
Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.
1Plant Molecular and Cellular Biology Program, University of Florida, 2Department of Biology, University of Florida, 3Interdisciplinary Center for Biotechnology Research, University of Florida, 4Genetics Institute, University of Florida
Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.
This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.
This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.
Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
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.
PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.
We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.
Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR
Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.
1Section on Neuronal Structure, Laboratory for Integrative Neuroscience, NIAAA, NIH, 2Department Physiology and Pharmacology, Wake Forest University Health Sciences, 3Oregon National Primate Research Center, Division of Neuroscience, Oregon Health and Science University
We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.
A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.
This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.
One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures
The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.
Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays
1Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health canada, 2National Institute for the Control of Pharmaceutical and Biological Products, The State Food and Drug Administration, Beijing, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 4Microbiology Department, Faculty of Medicine, King Abdulaziz University, 5National Microbiology Laboratory, Public Health Agency of Canada
A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.
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.
Advantages of the Trans-Blot® Turbo™ Transfer System Over Traditional Wet Tank and Semi-Dry Transfer Methods - ADVERTISEMENT
A demonstration of the advantages in speed, ease of use, and transfer efficiency of the Trans-Blot Turbo transfer system compared to conventional wet tank and semi-dry transfer methods.
γ-Herpesviruses (γ-HVs) establish life-long persistency in their host. Infection of mice with γ-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of γHVs. This protocol describes the detection and quantitation of γHV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.
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.
Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.
1Department of Mechanical Engineering, Texas A&M University, 2Department of Mechanical Engineering and Department of Nuclear Engineering, Texas A&M University, 3Department of Chemical Engineering, Texas A&M University
We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.
Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
1Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 2Department of Surgery, University of Toronto, 3Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill
The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.
Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.
Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1β using a Whole Blood Stimulation Assay
1Translational Immunology Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, 2Translational Autoinflammatory Disease Section, Office of the Clinical Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases
We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
The accuracy and sensitivity of protein determination by the rapid and convenient Bradford assay is compromised by intrinsic nonlinearity. We show a simple linearization procedure that greatly increases the accuracy, improves the sensitivity of the assay about 10-fold, and significantly reduces interference by detergents.
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.
Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography
We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.
Department of Medicine, St. Luke's Roosevelt Medical Center
Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.
High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors
In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.
This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles
We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.