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Mass Spectrometry: An analytical method used in determining the identity of a chemical based on its mass using mass analyzers/mass spectrometers.

Tandem Mass Spectrometry

JoVE 5690

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help elucidate its composition and sequence. This is accomplished by having mass spectrometers in series. The first spectrometer ionizes a sample and filter ions of a specific mass to charge ratio. Filtered ions are then fragmented and passed to a second mass spectrometer where the fragments are analyzed. This video introduces the principles of tandem mass spectrometry, including mass-to-ratio selection and dissociation methods. Also shown is a general procedure for analyzing a biochemical compound using tandem mass spectrometry with collision-induced dissociation. The applications section covers selection reaction monitoring, determination of protein post-translation modifications, and detection of tacrolimus levels in blood. Tandem mass spectrometry links together multiple stages of mass spectrometry to first isolate a biomolecule, and then determine aspects of its chemical makeup. Biomolecules have large, complex structures, making it difficult to determine their molecular composition. Tandem mass spectrometry selects a molecule of interest that is later fragmented into multiple subunits, which can help elucidate its identification and sequence. This video will show the


 Biochemistry

MALDI-TOF Mass Spectrometry

JoVE 5691

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing compounds in the gaseous state, samples are embedded in a matrix, which is struck by a laser. The matrix absorbs the majority of the energy; some of this energy is then transferred to the sample, which ionizes as a result. Sample ions can then be identified using a time-of-flight analyzer (TOF). This video covers principles of MALDI-TOF, including matrix selection and how TOF is used to elucidate mass-to-charge ratios. This procedure shows the preparation of a MALDI plate, the loading of samples onto the plate, and the operation of the TOF-mass spectrometer. In the final section, applications and variations are shown, including whole-cell analysis, characterization of complex biological samples, and electron spray ionization. Matrix-assisted laser desorption ionization, or MALDI, is a mass spectrometry ion source ideal for the analysis of biomolecules. Most ion sources remove structural information from large, fragile biomolecules. MALDI maintains structural integrity, and therefore information, while accelerating the molecules into the mass analyzer, which separates the compounds based on size and charge. The most commonly coupled with MALDI is the time of flight, or TOF,


 Biochemistry

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

Introduction to Mass Spectrometry

JoVE 5634

Source: Laboratory of Dr. Khuloud Al-Jamal - King's College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.

A mass spectrometer is composed of an ionization source, an analyzer, and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can b


 Analytical Chemistry

SNP Genotyping

JoVE 5544

Single nucleotide polymorphisms, or SNPs, are the most common form of genetic variation in humans. These differences at individual bases in the DNA often do not directly affect gene expression, but in many cases can still be useful for locating disease-associated genes or for diagnosing patients. Numerous methodologies have been established to identify, or “genotype”, SNPs.JoVE’s introduction to SNP Genotyping begins by discussing what SNPs are and how they can be used to identify disease-associated genes. Several common SNP genotyping methods are then examined, including direct hybridization, PCR-based methods, fragment analysis, and sequencing. Finally, we present several examples of how these techniques are applied to genetic research today.


 Genetics

Metabolic Labeling

JoVE 5687

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR. This video introduces the concepts of metabolic labeling and show two general procedures.  The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos. Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications. Metabolic lab


 Biochemistry

Internal Standards

JoVE 10225

Source: Laboratory of Dr. B. Jill Venton - University of Virginia

The goal of many chemical analyses is a quantitative analysis, where the amount of a substance in a sample is determined. In order to accurately calculate the concentration of an unknown from a sample, careful sample preparation is key. Every time a sample is handled or transferred, some of the sample can be lost. There are strategies however, for minimizing sample loss. There are also strategies for coping with sample loss and still making accurate measurements of concentration. To minimize sample loss, the ideal is to minimize the number of sample handling and transfer steps. For example, massing a solid sample directly into a flask that a solution will be made in reduces a transfer step. If it's necessary to transfer from one flask to another and a dilution is being made, then triple rinsing the glassware helps ensure all the sample is transferred. Other strategies are more specific to the sample. For example, samples that adsorb to glass, such as proteins, might better be handled in polypropylene disposable tubes. The tubes are not hydrophilic, so if a small amount of sample is to be pipetted in water, it is best to have already added the water to the tube, so the sample can be pipetted directly into the solve


 Analytical Chemistry

Gas Chromatography (GC) with Flame-Ionization Detection

JoVE 10187

Source: Laboratory of Dr. B. Jill Venton - University of Virginia

Gas chromatography (GC) is used to separate and detect small molecular weight compounds in the gas phase. The sample is either a gas or a liquid that is vaporized in the injection port. Typically, the compounds analyzed are less than 1,000 Da, because it is difficult to vaporize larger compounds. GC is popular for environmental monitoring and industrial applications because it is very reliable and can be run nearly continuously. GC is typically used in applications where small, volatile molecules are detected and with non-aqueous solutions. Liquid chromatography is more popular for measurements in aqueous samples and can be used to study larger molecules, because the molecules do not need to vaporize. GC is favored for nonpolar molecules while LC is more common for separating polar analytes. The mobile phase for gas chromatography is a carrier gas, typically helium because of its low molecular weight and being chemically inert. Pressure is applied and the mobile phase moves the analyte through the column. The separation is accomplished using a column coated with a stationary phase. Open tubular capillary columns are the most popular columns and have the stationary phase coated on the walls of the capillary. Stationary phases a


 Analytical Chemistry

Electric Charge in a Magnetic Field

JoVE 10133

Source: Andrew Duffy, PhD, Department of Physics, Boston University, Boston, MA

This experiment duplicates J.J. Thomson's famous experiment at the end of the 19th century, in which he measured the charge-to-mass ratio of the electron. In combination with Robert A. Millikan's oil-drop experiment a few years later that produced a value for the charge of the electron, the experiments enabled scientists to find, for the first time, both the mass and the charge of the electron, which are key parameters for the electron. Thomson was not able to measure the electron charge or the electron mass separately, but he was able to find their ratio. The same is true for this demonstration; although here there is the advantage of being able to look up the values for the magnitude of the charge on the electron(e) and the mass of the electron (me), which are now both known precisely.


 Physics II

An Introduction to Cell Metabolism

JoVE 5652

In cells, critical molecules are either built by joining together individual units like amino acids or nucleotides, or broken down into smaller components. Respectively, the reactions responsible for this are referred to as anabolic and catabolic. These reactions require or produce energy typically in the form of a “high-energy” molecule called ATP. Together, these processes make up “Cell Metabolism,” and are hallmarks of healthy, living cells.JoVE’s introduction to cell metabolism briefly reviews the rich history of this field, ranging from early studies on photosynthesis to more recent discoveries pertaining to energy production in all cells. This is followed by a discussion of some key questions asked by scientists studying metabolism, and common methods that they apply to answer these questions. Finally, we’ll explore how current researchers are studying alterations in metabolism that accompany metabolic disorders, or that occur following exposure to environmental stressors.


 Cell Biology

DNA Methylation Analysis

JoVE 5550

Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the methylation of clusters of methylation sites, called “CpG islands”, near promoters and other gene regulatory elements may contribute to the stable silencing of genes, for example, during epigenetic processes such as genomic imprinting and X-chromosome inactivation. At the same time, aberrant CpG methylation has been shown to be associated with cancer.In this video, the biological functions and mechanisms of DNA methylation will be presented, along with various techniques used to identify methylation sites in the genome. We will then examine the steps of bisulfite analysis, one of the most commonly used methods for detecting DNA methylation, as well as several applications of this technique.


 Genetics

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

1Elemental Bio-imaging Facility, University of Technology Sydney, 2Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 3Department of Pathology, The University of Melbourne, 4School of Earth Sciences, The University of Melbourne, 5Research School, Ruhr University, 6Department of Physiology, Monash University, 7ESI Ltd., Bozeman, 8Agilent Technologies, Mulgrave

JoVE 55042


 Medicine

Performing 1D Thin Layer Chromatography

JoVE 5499

Source: Laboratory of Dr. Yuri Bolshan — University of Ontario Institute of Technology

Thin layer chromatography (TLC) is a chromatographic method used to separate mixtures of non-volatile compounds. A TLC plate consists of a thin layer of adsorbent material (the stationary phase) fixed to an appropriate solid support such as plastic, aluminum, or glass1. The sample(s) and reference compound(s) are dissolved in an appropriate solvent and applied near the bottom edge of the TLC plate in small spots. The TLC plate is developed by immersing the bottom edge in the developing solvent consisting of an appropriate mobile phase. Capillary action allows the mobile phase to move up the adsorbent layer. As the solvent moves up the TLC plate, it carries with it the components of each spot and separates them based on their physical interactions with the mobile and stationary phases.


 Organic Chemistry

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, 4The State Key Laboratory of Medical Genetics, Central South University

JoVE 56739


 Cancer Research

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

1Molecular Genetics of Chronic Inflammation and Allergic Disease, Max-Delbrück Center for Molecular Medicine, 2Murdoch Childrens Research Institute, 3Department of Paediatrics, University of Melbourne, 4Centre for Social and Early Emotional Development, Faculty of Health, Deakin University, 5Department of Paediatrics, University of Western Australia

JoVE 57601


 Genetics

Recombineering and Gene Targeting

JoVE 5553

One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based on stretches of similar or identical sequences. Together with gene targeting, which takes advantage of endogenous HR to alter an organism’s genome at a specific loci, HR-based cloning techniques have greatly improved the speed and efficacy of high-throughput genetic engineering.In this video, we introduce the principles of HR, as well as the basic components required to perform a recombineering experiment, including recombination-competent organisms and genomic libraries such as bacterial artificial chromosomes (BAC). We then walk through a protocol that uses recombineering to generate a gene-targeting vector that can ultimately be transfected into embryonic stem cells to generate a transgenic animal. Finally, several applications that highlight the utility and variety of recombineering techniques wi


 Genetics

Quantitative Analysis of Chromatin Proteomes in Disease

1Department of Anesthesiology, David Geffen School of Medicine at UCLA, 2Department of Medicine, David Geffen School of Medicine at UCLA, 3Department of Physiology, David Geffen School of Medicine at UCLA, 4Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah

JoVE 4294


 Medicine

Lead Analysis of Soil Using Atomic Absorption Spectroscopy

JoVE 10021

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Lead occurs naturally in soil, in levels ranging from 10-50 ppm. However, with the widespread use of lead in paint and gasoline in addition to contamination by industry, urban soils often have concentrations of lead significantly greater than background levels – up to 10,000 ppm in some places. Ongoing problems arise from the fact that lead does not biodegrade, and instead remains in the soil. Serious health risks are associated with lead poisoning, where children are particularly at risk. Millions of children in the U.S. are exposed to soil containing lead. This exposure can cause developmental and behavioral problems in children. These problems include learning disabilities, inattention, delayed growth, and brain damage. The Environmental Protection Agency has set a standard for lead in soil at 400 ppm for play areas and 1,200 ppm for non-play areas. Lead is also of concern in soil, when it’s used for gardening. Plants take up lead from the soil. Therefore, vegetables or herbs grown in contaminated soil can lead to lead poisoning. In addition, contaminated soil particles can be breathed in while gardening or brought into the house on clothing and footwear. It is recommended that s


 Environmental Science

Proper Operation of Vacuum Based Equipment

JoVE 10377

Source: Robert M. Rioux, Ajay Sathe & Zhifeng Chen, Pennsylvania State University, University Park, PA

Vacuum is required for a number of laboratory procedures. This is most routinely achieved in the laboratory by the use of vacuum pumps. In addition to working at low pressures, vacuum pumps can also be used to enable rapid changing of the atmospheres in a reactor or flask by evacuation and backfilling.


 Lab Safety

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

1Graduate Program in Cellular and Molecular Pharmacology, School of Graduate Studies, Rutgers University, The State University of New Jersey, 2Graduate Program in Quantitative Biomedicine, School of Graduate Studies, Rutgers University, The State University of New Jersey, 3Department of Medicine, Division of Medical Oncology, Rutgers Robert Wood Johnson Medical School, 4Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, UCLA Metabolomics Center, and California NanoSystems Institute, David Geffen School of Medicine, University of California, Los Angeles, 5Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, 6Pharmacology, Rutgers Robert Wood Johnson Medical School, 7Cancer Metabolism and Growth Program, Rutgers Cancer Institute of New Jersey

JoVE 57996


 Cancer Research

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