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Sodium Dodecyl Sulfate: An anionic surfactant, usually a mixture of sodium alkyl sulfates, mainly the lauryl; lowers surface tension of aqueous solutions; used as fat emulsifier, wetting agent, detergent in cosmetics, pharmaceuticals and toothpastes; also as research tool in protein biochemistry.

Separating Protein with SDS-PAGE

JoVE 5058

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions. This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.


 Basic Methods in Cellular and Molecular Biology

Two-Dimensional Gel Electrophoresis

JoVE 5686

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights. The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM). Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially a


 Biochemistry

Community DNA Extraction from Bacterial Colonies

JoVE 10218

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner

Traditional methods of analysis for microbial communities within soils have usually involved either cultural assays utilizing dilution and plating methodology on selective and differential media or direct count assays. Direct counts offer information about the total number of bacteria present, but give no information about the number or diversity of populations present within the community. Plate counts allow enumeration of total cultural or selected cultural populations, and hence provide information on the different populations present. However, since less than 1% of soil bacteria are readily culturable, cultural information offers only a piece of the picture. The actual fraction of the community that can be cultured depends on the medium chosen for cultural counts. Any single medium will select for the populations that are best suited to that particular medium. In recent years, the advantages of studying community DNA extracted from soil samples have become apparent. This nonculture-based approach is thought to be more representative of the actual community present than culture-based approaches. In addition to providing information about the types of populations present, this


 Environmental Microbiology

Plasmid Purification

JoVE 5062

Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification. Plasmid purification includes three basic steps: growth of the bacterial culture, harvesting and lysis of the bacteria, and purification of the plasmid DNA. The video contains an explanation where the plasmid can be found in each step of the protocol and to quantitatively and qualitatively analyze plasmid DNA with a spectrophotometer and/or gel electrophoresis. There are different types of plasmid purification methods available, which are geared toward desired yield, plasmid copy number, and bacterial culture volume.


 Basic Methods in Cellular and Molecular Biology

Isolating Nucleic Acids from Yeast

JoVE 5096

One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.

This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps common to the isolation of all biomacromolecules. Next, we will discuss two unique purification methods for the separation of nucleic acids: column binding and phase separation. Additionally, we will demonstrate several ways in which these methods are applied in the laboratory, including the preparation of nucleic acids for molecular biology techniques such as PCR and southern blotting, quantification of gene expression in response to environmental stimuli, and purification of large amounts of recombinant proteins.


 Biology I

The Western Blot

JoVE 5065

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


 Basic Methods in Cellular and Molecular Biology

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

1College of Chemistry and Molecular Engineering, Peking University, 2Beijing National Laboratory for Molecular Sciences, Peking University, 3Peking-Tsinghua Center for Life Sciences, Peking University, 4Synthetic and Functional Biomolecules Center, Peking University, 5Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University

JoVE 58580


 Biochemistry

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

1Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, Feinberg School of Medicine, Northwestern University, 3Department of Biomedical Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, 5Department of Internal Medicine, University of New Mexico HSC, 6Department of Pathology, University of New Mexico HSC, 7Department of Chemical and Biological Engineering, Northwestern University, 8Chemistry of Life Processes Institute, Northwestern University, 9Department of Surgery, Jesse Brown VA Medical Center

JoVE 53271


 Bioengineering

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants As 3D-like Platforms for in Vitro Studies

1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, 4Gladstone Institutes, University of California San Francisco, 5Faculty of Medicine, University of Porto

Video Coming Soon

JoVE 56924


 JoVE In-Press

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

1Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), 2Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 3School of Biological, Biomedical & Environmental Sciences, University of Hull

JoVE 54230


 Cancer Research

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

1Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 2Center for Measurement Standards, Industrial Technology Research Institute, 3National Synchrotron Radiation Research Center, 4Department of Chemistry, National Changhua University of Education

JoVE 53500


 Bioengineering

Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model - Introduction of the Surgical Technique

1Department of Neurosurgery, Kantonsspital Aarau, 2Neuro Lab, Research Group for Experimental Neurosurgery and Neurocritical Care, Department of Intensive Care Medicine, University Hospital and University of Bern, 3Division of Neuroradiology, Department of Radiology, Kantonsspital Aarau

JoVE 56359


 Neuroscience

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

1Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 2Department of Biological Sciences, Texas Tech University, 3CISER (Center for the Integration of STEM Education & Research), Texas Tech University

JoVE 57600


 Biochemistry

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