Source: Vy M. Dong and Daniel Kim, Department of Chemistry, University of California, Irvine, CA
Controlling the reactivity and selectivity during the synthesis of a molecule is very important criteria for chemists. This has led to the development of many reagents that allow chemists to pick and choose reagents suitable for a given task. Quite often, a balance between reactivity and selectivity needs to be achieved. This experiment will use IR spectroscopy to monitor the reaction and to understand the reactivity of carbonyl compounds as well as the reactivity of hydride-reducing reagents.…
Organic Chemistry II
A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or “recycled” back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.…
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…
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
Source: Roberto Leon, Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA
Concrete is one of the most common construction materials and consists of two phases: the mortar phase, comprised of concrete, water and air, and the aggregate phase, comprised of coarse and fine aggregates. There are two key considerations when designing a concrete mix. First, the concrete must be workable and easy to cast in the forms in its fresh condition, even when the forms are packed with steel reinforcement. In this condition, it is the rheology of concrete that is important. Second, the mix must produce a hardened concrete of specified strength at 28 days (or similar specified time) that is durable and provides good serviceability.
In this laboratory exercise, a method of concrete mixture proportioning, named the trial batch method, will be explored. The concrete produced will be used in conducting typical tests to determine the principal characteristics of fresh concrete, including slump, flowability, air content, and density. The trial batch method is a simple, empirical approach to mixture design.
The objectives of this experiment are fourfold: (1) to use the trial batch mix method to determine optimum proportions of aggregates, cement, an…
Source: Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Through auscultation, the clinician is able "to eavesdrop on the workings of the body" to gain important diagnostic information.1 Historically, the term "auscultation" was synonymous with "immediate auscultation," in which the examiner's ear was placed directly against the patient's skin. Although this was standard practice for centuries, the method proved inadequate in nineteenth-century France, due to social norms and suboptimal diagnostic yield. This led René Laënnec to invent the first stethoscope in 1816 (Figure 1), a tool that has since become inseparable from auscultation in modern clinical practice, and patients hold it as a symbol of honor and trustworthiness among those who carry them.2
Figure 1. A representative illustration of the first stethoscope invented by René Laënnec.
The stethoscope has undergone many technologic advances since Laënnec's initial hollow wooden tube. Practically speaking, the provider mus…
Physical Examinations I
Robert M. Rioux & Zhifeng Chen, Pennsylvania State University, University Park, PA
The most common laboratory emergencies include chemical spills, fire or explosion, electric shock, and personnel injuries. Most laboratory accidents occur due to poor planning or lack of attention. Therefore, it's always better to prevent accidents (being proactive) than having to take any actions during an emergency (being reactive). For example, always wear proper personal protective equipment (PPE) in the laboratory. Regular laboratory inspection and equipment maintenance is beneficial to prevent laboratory accidents. However, once the emergency occurs, it's also essential to know what to do. Ensure your personal safety first and then call local emergency responders, when and if necessary. The extent of your response will depend on the seriousness of the incident and documented laboratory protocols for dealing with such incidents. Stay calm and take proper actions according to the type and level of emergency.…
Source: Robert M. Rioux & Taslima A. Zaman, Pennsylvania State University, University Park, PA
While the use of various chemicals in experimental research is essential, it is also important to safely store and maintain them as a part of the Environmental, Health and Safety (EHS) program. The properties of chemicals and their reactivity vary broadly and if chemicals are not managed, stored, and labeled properly, they can have harmful or even destructive consequences such as toxic fume production, fire or explosion, which may result in human fatality, property damage or environmental hazards. Therefore, an appropriate chemical label should identify the material and list the associated hazards, and users should have knowledge of how to read chemical labels and safety data sheets (SDS). Proper chemical storage must meet OSHA (Occupational Safety and Health Association) standards and this can prevent most chemical reactivity hazards.…
Source: Laboratory of Dr. Ana J. García-Sáez — University of Tübingen
Most samples of interest are mixtures of many different components. Sample preparation, a key step in the analytical process, removes interferences that may affect the analysis. As such, developing separation techniques is an important endeavor not just in academia, but also in industry.
One way to separate mixtures is to use their solubility properties. In this short paper, we will deal with aqueous solutions. The solubility of a compound of interest depends on (1) ionic strength of solution, (2) pH, and (3) temperature. By manipulating with these three factors, a condition in which the compound is insoluble can be used to remove the compound of interest from the rest of the sample.1…
Source: Laboratory of Dr. Lynne O'Connell — Boston College
When the conditions of a system at equilibrium are altered, the system responds in such a way as to maintain the equilibrium. In 1888, Henri-Lewis Le Châtelier described this phenomenon in a principle that states, "When a change in temperature, pressure, or concentration disturbs a system in chemical equilibrium, the change will be counteracted by an alteration in the equilibrium composition."
This experiment demonstrates Le Châtelier's principle at work in a reversible reaction between iron(III) ion and thiocyanate ion, which produces iron(III) thiocyante ion:
Fe3+(aq) + SCN- (aq) FeSCN2+ (aq)
The concentration of one of the ions is altered either by directly adding a quantity of one ion to the solution or by selectively removing an ion from the solution through formation of an insoluble salt. Observations of color changes indicate whether the equilibrium has shifted to favor formation of the products or the reactants. In addition, the effect of a temperature change on the solution at equilibrium can be obs…
Source: Hsin-Chun Chiu and Tyler J. Morin, laboratory of Dr. Ian Tonks—University of Minnesota Twin Cities
Schlenk lines and high vacuum lines are both used to exclude moisture and oxygen from reactions by running reactions under a slight overpressure of inert gas (usually N2 or Ar) or under vacuum. Vacuum transfer has been developed as a method separate solvents (other volatile reagents) from drying agents (or other nonvolatile agents) and dispense them to reaction or storage vessels while maintaining an air-free environment. Similar to thermal distillations, vacuum transfer separates solvents by vaporizing and condensing them in another receiving vessel; however, vacuum transfers utilize the low pressure in the manifolds of Schlenk and high vacuum lines to lower boiling points to room temperature or below, allowing for cryogenic distillations. This technique can provide a safer alternative to thermal distillation for the collection of air- and moisture-free solvents. After the vacuum transfer, the water content of the collected solvent can be tested quantitatively by Karl Fischer titration, qualitatively by titration with a Na/Ph2CO solution, or by 1H NMR spectroscopy.…