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Förster Resonance Energy Transfer (FRET)

JoVE 5696

Förster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent molecule can non-radiatively transfer energy to an acceptor molecule if their respective emission and absorbance spectra overlap. The amount of energy transferred—and consequently the overall emission of sample—depends on the proximity of an acceptor-donor pair of photoluminescent molecules. FRET analysis is combined with other biochemistry techniques to obtain detailed information of biomolecular structures and interactions from this “spectroscopic ruler.” This video covers the principles and concepts of FRET analysis. The procedure focuses on preparing samples for FRET and ways to present and interpret data. Finally, the applications include monitoring conformational and cellular processes by labeling parts of a cell or protein, monitoring enzyme reactions that alter protein structures, and using FRET to monitor aggregation of monomers expressed by cells. Förster Resonance Energy Transfer, or FRET, is a non-radiative transfer of energy between light-emitting molecules, and is often used to investigate close-range biochemical interactions. FRET only occurs when fluorescent molecules are spaced within 10 nm of each other. FRET analysis


 Biochemistry

Surface Plasmon Resonance (SPR)

JoVE 5697

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, specificity, and concentration of biomolecules. In SPR, biomolecular interactions occur on a biosensor made of a thin layer of metal on a prism. Real-time interactions of biomolecules can be monitored by measuring the changes of light reflected off the underside of the metal. This video describes the basic concepts of SPR and how it is used to analyze and visualize biomolecular interactions. This is followed by a sample preparation and experimental protocol for investigating binding rates using SPR. In the applications section, SPR imaging, localized SPR, and quantum dot enhanced SPR are explored. Surface plasmon resonance, or SPR, is the underlying phenomenon behind certain label-free biosensors for evaluating binding and adsorption interactions of biomolecules. Binding assays that require labeling, such as ELISA, can be a time-consuming process, and may alter the functionality of the analyte. In SPR, biomolecular interactions occur on a special sensor made of a thin layer of metal on one face of a prism. By monitoring the changes in light reflected off of the underside of the metal, SPR instruments visualize these interactions in real-time without the use of labels. This video


 Biochemistry

Electrochemical Measurements of Supported Catalysts Using a Potentiostat/Galvanostat

JoVE 5698

Source: Laboratory of Dr. Yuriy Román — Massachusetts Institute of Technology

A potentiostat/galvanostat (often referred to as simply a potentiostat) is an instrument that measures current at an applied potential (potentiostatic operation) or measures potential at an applied current (galvanostatic operation) (Figure 1). It is the most commonly used instrument in the electrochemical characterization of anode and cathode materials for fuel cells, electrolyzers, batteries, and supercapacitors. Conventionally, these anode and cathode materials are interfaced with a potentiostat via a three-electrode electrochemical cell. The electrode leads from the potentiostat are connected to the reference electrode, the counter electrode (often called the auxiliary electrode), and the working electrode (which contains the test material of interest). The electrochemical cell is then filled with a high ionic strength electrolyte solution, such as an acidic, alkaline, or salt solution. The media for this high ionic strength solution is typically aqueous; however, for applications necessitating higher operating cell potential windows, such as batteries and supercapacitors, non-aqueous media is often used. The cell media is degassed with an inert gas (to prevent unwanted side react


 Analytical Chemistry

Introduction to Titration

JoVE 5699

Source: Laboratory of Dr. Yee Nee Tan — Agency for Science, Technology, and Research

Titration is a common technique used to quantitatively determine the unknown concentration of an identified analyte.1-4 It is also called volumetric analysis, as the measurement of volumes is critical in titration. There are many types of titrations based on the types of reactions they exploit. The most common types are acid-base titrations and redox titrations.5-11 In a typical titration process, a standard solution of titrant in a burette is gradually applied to react with an analyte with an unknown concentration in an Erlenmeyer flask. For acid-base titration, a pH indicator is usually added in the analyte solution to indicate the endpoint of titration.12 Instead of adding pH indicators, pH can also be monitored using a pH meter during a titration process and the endpoint is determined graphically from a pH titration curve. The volume of titrant recorded at the endpoint can be used to calculate the concentration of the analyte based on the reaction stoichiometry. For the acid-base titration presented in this video, the titrant is a standardized sodium hydroxide solution and the analyte is domestic vinegar. Vinegar is an acidic liquid that


 General Chemistry

Igneous Intrusive Rock

JoVE 10036

Source: Laboratory of Alan Lester - University of Colorado Boulder

Igneous rocks are products of the cooling and crystallization of high temperature liquid rock, called magma. Magmatic temperatures typically range from approximately 800 °C to 1,200 °C. Molten rock is, perhaps luckily for humans, an anomaly on planet Earth. If a random and imaginary drill hole were made in the Earth, it would most likely not reach a region of truly and totally molten material until the outer core, at nearly 2,900 km beneath the surface (Earth's radius is 6,370 km). Even there, this molten material would predominantly consist of liquid iron, not true silicate rock, and be incapable of ever reaching Earth's surface. Volcanic eruptions and igneous rocks do occur though, and they are evidence that there are indeed isolated regions of melting and magma generation within the Earth.


 Earth Science

Nutrients in Aquatic Ecosystems

JoVE 10023

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

Nitrogen and phosphorus are essential plant nutrients found in aquatic ecosystems and both are monitored as a part of water quality testing because in excess amounts they can cause significant water quality problems. 

Nitrogen in water is measured as the common form nitrate (NO3-) that is dissolved in water and readily absorbed by photosynthesizers such as algae. The common form of phosphorus measured is phosphate (PO43-), which is strongly attracted to sediment particles as well as dissolved in water. In excess amounts, both nutrients can cause an increase in aquatic plant growth (algal bloom, Figure 1) that can disrupt the light, temperature, and oxygen levels in the water below and lead to eutrophication and hypoxia (low dissolved oxygen in water) forming a “dead zone” of no biological activity. Sources of nitrates and phosphorus include wastewater treatment plants, runoff from fertilized lawns and agricultural lands, faulty septic systems, animal manure runoff, and industrial waste discharge. Figu


 Environmental Science

Measuring Tropospheric Ozone

JoVE 10024

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

Ozone is a form of elemental oxygen (O3), a molecule of three oxygen atoms bonded in a structure that is highly reactive as an oxidizing agent. Ozone occurs in both the stratosphere and the troposphere levels of the atmosphere. When in the stratosphere (located approximately 10-50 km from the earth’s surface), ozone molecules form to the ozone layer and help prevent harmful UV rays from reaching Earth’s surface. In lower altitudes of the troposphere (surface - approximately 17 km), ozone is harmful to human health and is considered an air pollutant contributing to photochemical smog (Figure 1). Ozone molecules can cause damage directly by harming respiratory tissue when inhaled or indirectly by harming plant tissues (Figure 2) and softer materials including tires on automobiles. Outdoor tropospheric ozone is formed at ground level when nitrogen oxides (NOx) and volatile organic compounds (VOCs) from automobile emissions are exposed to sunlight. Consequently, health concerns over ozone concentrations escalate in sunny conditions or when and where automobile use is increased. Reaction: NO2 + VOC + sunlight &


 Environmental Science

Igneous Volcanic Rock

JoVE 10037

Source: Laboratory of Alan Lester - University of Colorado Boulder

Igneous rocks are the products of cooling and crystallization of magma. Volcanic rocks are a particular variety of igneous rock, forming as a consequence of magma breaching the surface, then cooling and crystallizing in the subaerial environment. 

Magma is liquid rock that typically ranges in temperature from approximately 800 °C to 1,200 °C (Figure 1). Magma itself is produced within the Earth via three primary melting mechanisms, namely the addition of heat, addition of volatiles, and decompression. Each mode of melt generation tends to produce specific types of magma and, therefore, distinct eruptive styles and structures. Figure 1. Fresh lava breakout on Kilauea, Hawaii. Lava is the term for magma that is on Earth’s surface.


 Earth Science

Determination of Moisture Content in Soil

JoVE 10011

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

Soils normally contain a finite amount of water, which can be expressed as the “soil moisture content.” This moisture exists within the pore spaces in between soil aggregates (inter-aggregate pore space) and within soil aggregates (intra-aggregate pore space) (Figure 1). Normally this pore space is occupied by air and/or water. If all the pores are occupied by air, the soil is completely dry. If all the pores are filled with water, the soil is said to be saturated. Figure 1. Pore space in soil.


 Environmental Microbiology

Visualizing Soil Microorganisms via the Contact Slide Assay and Microscopy

JoVE 10053

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

Soil comprises the thin layer at the earth’s surface, containing biotic and abiotic factors that contribute to life. The abiotic portion includes inorganic particles ranging in size and shape that determine the soil’s texture. The biotic portion incorporates plant residues, roots, organic matter, and microorganisms. Soil microbe abundance and diversity is expansive, as one gram of soil contains 107-8 bacteria, 106-8 actinomycetes, 105-6 fungi, 103 yeast, 104-6 protozoa, 103-4 algae, and 53 nematodes. Together, the biotic and abiotic factors form architectures around plant roots, known as the rhizosphere, that provide favorable conditions for soil microorganisms. Biotic and abiotic factors promote life in soils. However, they also contribute stressful dynamics that limit microbes. Biotic stress involves competition amongst life to adapt and survive in environmental conditions. For example, microbes can secrete inhibitory or toxic substances to harm neighboring microorganisms. Penicillium notatum is a notorious fungus, as it reduces competition for nutrients by producing an a


 Environmental Microbiology

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