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 JoVE Biology

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


 JoVE Bioengineering

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

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


 JoVE Immunology and Infection

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

1Department of Microbiology, Biochemistry, Immunology, Morehouse School of Medicine, 2Department of Medicine, Emory University School of Medicine, 3Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 4Yerkes National Primate Research Center


 JoVE Bioengineering

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


 JoVE Medicine

Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein

1Neurodegenerative Diseases Unit, ANSES - French Agency for Food, Environmental and Occupational Health & Safety, 2Indicia Production, 3AJ Roboscreen GmbH, 4Epidemiology Unit, ANSES - French Agency for Food, Environmental and Occupational Health & Safety, 5Experimental Facilities, ANSES - French Agency for Food, Environmental and Occupational Health & Safety


 JoVE Biology

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

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


 JoVE Immunology and Infection

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.


 Science Education: Basic Methods in Cellular and Molecular Biology

The Western Blot

JoVE Science Education

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry. This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through severa

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