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.
A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents
1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Program in Molecular Medicine, University of Maryland School of Medicine, 3Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, 4Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine, 5Department of Pathology and Biochemistry & Molecular Biology, University of Maryland School of Medicine
We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.
Published August 31, 2013. Keywords: Cellular Biology, Transcription Factors, Vitamin D, Drug Discovery, Enzyme-Linked Immunosorbent Assay (ELISA), DNA-binding, transcription factor, drug screening, antibody
Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments
1Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.
Published July 5, 2014. Keywords: Biochemistry, two-electrode voltage-clamp, electrophysiology, biotinylation, Xenopus laevis oocytes, epithelial sodium channel, ENaC, proteases, proteolytic channel activation, ion channel, cleavage sites, cleavage fragments
V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting
V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.
Published December 30, 2013. Keywords: Basic Protocol, Biotechnology, Pharmaceutical, Protein electrophoresis, Western blot, Stain-Free, loading control, total protein normalization, stain-free technology
1Department of Neuroscience, University of Minnesota, 2Institute for Translational Neuroscience, University of Minnesota, 3Department of Obstetrics, Gynecology, and Women’s Heath, University of Minnesota, 4Masonic Cancer Center, University of Minnesota
The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.
1Fraunhofer USA Center for Molecular Biotechnology
Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.
Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo
1Molekulare Mikrobiologie, Universität Osnabrück
A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.
1Center for Biotechnology Education, Krieger School of Arts and Sciences, Johns Hopkins University, 2Qiagen Sciences, Inc.
Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.
Published August 22, 2013. Keywords: Microbiology, Genetics, Molecular Biology, Basic Protocols, Genomics, Eukaryota, Bacteria, Viruses, Bacterial Infections and Mycoses, Virus Diseases, Diagnosis, Therapeutics, Equipment and Supplies, Technology, Industry, and Agriculture, Life Sciences (General), Pyrosequencing, DNA, Microbe, PCR, primers, Next-Generation, high-throughput, sequencing
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
1Department of Pathology, New York University School of Medicine, 2Division of Infectious Diseases, Department of Medicine, Mount Sinai Medical Center, 3Division of Hematology and Oncology, Hess Center for Science and Medicine, Mount Sinai Medical Center
Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1β expression.
1Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, 2Department of Radiation Oncology, Stanford School of Medicine, 3Service Hospitalier Frédéric Joliot, Institut d'Imagerie Biomédicale, 4Vanderbilt School of Medicine, 5The Walter & Eliza Hall Institute of Medical Research, 6Unité INSERM U786, Institut Pasteur, 7Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur
Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.
Published May 23, 2014. Keywords: Bioengineering, Biochemical Phenomena, Biochemical Processes, Energy Transfer, Fluorescence Resonance Energy Transfer (FRET), FUEL, BRET, CRET, Förster, bioluminescence, In vivo
1Center for Health Sciences, SRI International, 2Department of Chemistry and Biochemistry, University of California-Santa Cruz
Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration. A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.
Published January 7, 2014. Keywords: Medicine, MPTP, dopamine, Iba1, TH, GFAP, lipoxygenase, transgenic, gene-environment interactions, mouse, Parkinson's disease, neurodegeneration, neuroinflammation
DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems
1Physical Biosciences Division, Lawrence Berkeley National Laboratory
This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.
Published July 21, 2014. Keywords: Genetics, DNA-Affinity-Purified-chip, response regulator, transcription factor binding site, two component system, signal transduction, Desulfovibrio, lactate utilization regulator, ChIP-chip
1Lurie Family Imaging Center, Dana-Farber Cancer Institute, Harvard Medical School, 2Division of Pediatric Hematology/Oncology/Stem Cell Transplantation, Columbia University Medical Center
We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.
Published August 16, 2013. Keywords: Immunology, Infection, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Cancer Biology, Stem Cell Biology, Inflammation, Phagocytes, Phagocyte, Superoxides, Molecular Imaging, chemiluminescence, in vivo imaging, superoxide, bioluminescence, chronic inflammation, acute inflammation, phagocytes, cells, imaging, animal model
1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York
Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular “tags” for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.
Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species
Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.
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.
1Array Group, Assay Department, R&D Systems, Inc.
Proteome Profiler antibody arrays are a convenient and cost efficient way to screen for changes in receptor tyrosine kinase (RTK) phosphorylation without performing numerous immunoprecipitation (IP) Westerns. The ARY001 Human RTK array allows for the qualitative measurement of multiple RTKs in a single sample using chemiluminescence detection.
Published September 27, 2012. Keywords: Advertisement, Molecular Biology, Biochemistry, Cancer Biology, R & D Systems, Proteome Profiler, antibody array, receptor tyrosine kinase (RTK), phosphorylation, multiplexing, sandwich assay, chemiluminescence
The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.
A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems
1Department of Biotechnology, University of Verona, Verona, Italy, 2Department of Internal Medicine, University of Perugia, Perugia, Italy
In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.
Published March 23, 2015. Keywords: Plant Biology, Plant biotechnology, transient expression, stable expression, transgenic plant, Nicotiana tabacum, Nicotiana benthamiana, Baculovirus/insect cells, recombinant protein
Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)
The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.
Published February 18, 2013. Keywords: Neuroscience, Biochemistry, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Proteins, synapse, cultured hippocampal neurons, palmitoylation, lipid, immunoprecipitation, western blotting, biotin, Acyl-Biotin Exchange, ABE, neuron, brain, cell culture, rat, mouse, animal model
Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor
1Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 2Division of Regenerative Medicine, Tulane National Primate Research Center, 3Department of Microbiology and Immunology, Tulane University School of Medicine, 4Department of Pharmacology, Tulane University School of Medicine
Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
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.
1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University
We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.
Published September 13, 2013. Keywords: Genetics, Receptors, G-Protein-Coupled, Protein Engineering, Signal Transduction, Biochemistry, Unnatural amino acid, site-directed mutagenesis, G protein-coupled receptor, targeted photocrosslinking, bioorthogonal labeling, targeted epitope tagging
Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80
1Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health
This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.
1MRC Centre for Molecular Bacteriology and Infection, Division of Cell & Molecular Biology, Imperial College London, 2Preclinical Imaging, Caliper- A PerkinElmer Company
Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with μCT imaging to create a 4D movie of C. rodentium infection.
Published August 13, 2013. Keywords: Infection, Immunology, Cellular Biology, Molecular Biology, Microbiology, Genetics, Biophysics, Biomedical Engineering, Medicine, Anatomy, Physiology, Infectious Diseases, Bacterial Infections, Bioluminescence, DLIT-μCT, C. rodentium, 4D imaging, in vivo imaging, multi-modality imaging, CT, imaging, tomography, animal model
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.
Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response
1Department of Biological Sciences, University of Alabama
Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.
Published December 4, 2013. Keywords: Immunology, biology (general), environmental effects (biological, animal and plant), immunology, animal models, Immune System Diseases, Pathological Conditions, Signs and Symptoms, Life Sciences (General), Neuroinflammation, inflammation, nitric oxide, nitric oxide synthase, Drosophila, neurodegeneration, brain, Griess assay, nitrite detection, innate immunity, Parkinson disease, tissue culture
Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
1Lady Davis Institute for Medical Research, Jewish General Hospital, 2Department of Medicine, McGill University
Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.
Published December 3, 2014. Keywords: Biochemistry, RNA metabolism, mRNA translation, post-transcriptional gene regulation, mRNA stability, IRE, IRP1, IRP2, iron metabolism, ferritin, transferrin receptor
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.
Published March 1, 2013. Keywords: Biomedical Engineering, Bioengineering, Medicine, Biophysics, Materials Science, Physics, Pharmacology, Toxicology, Surgery, Chemistry and Materials (General), graphene, biomedical implants, surface modification, chemical vapor deposition, protein expression, confocal microscopy, implants, stents, clinical
1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen
A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.
Published December 17, 2012. Keywords: Plant Biology, Molecular Biology, Cellular Biology, Genetics, Genomics, Proteomics, Proteins, Cell Walls, Polysaccharides, Monoclonal Antibodies, Microarrays, CoMPP, glycans, Arabidopsis, tissue collection
1Department of Developmental Biology, Stanford University School of Medicine
Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.
1Department of Medicine, Vanderbilt University School of Medicine, 2Departments of Medicine and Immunology, Roswell Park Cancer Institute, 3Department of Medicine, University at Buffalo School of Medicine
NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.
This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.
Published July 3, 2013. Keywords: Cancer Biology, Medicine, Genetics, Cellular Biology, Molecular Biology, Biochemistry, Cancer Biology, Bioengineering, Genomics, Drug Discovery, RNA Interference, Cell Biology, Neoplasms, luciferase reporters, functional genomics, chemical biology, high-throughput screening technology, signal transduction, PCR, transfection, assay
A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor
1Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 2First department of Internal Medicine, School of Medicine, Kyorin University, 3Neosilk Laboratory, Immuno Biological Laboratories Co., Ltd.
We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.
1Department of Pathology, Case Western Reserve University School of Medicine, 2Cell Biology Program, Case Western Reserve University School of Medicine, 3Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine
To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.
Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast
Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.
SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval
1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University
We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.
This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.
Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.
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.
1Cell Signaling Technology, Inc.
Here we provide an extensive western blot troubleshooting guide based on our many years of experience, offering solutions to save you valuable time and reagents.