...Chromatography INTRODUCTION IN THIS LAB, WE WILL BE EXPRESSING THE GREEN FLUORESCENT PROTEIN (GFP) IN BACTERIA AND PURIFYING IT USING COLUMN CHROMATOGRAPHY. THE SPECIFIC TYPE OF CHROMATOGRAPHY WE WILL BE USING IS HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC). THE FOLLOWING IS INFORMATION FROM BIO-RAD INC., THE SUPPLIER OF THE REAGENTS: GFP has several stretches of hydrophobic amino acids, which results in the total protein being very hydrophobic. When the supernatant, rich in GFP, is passed over a HIC column in a highly salty buffer (Binding Buffer), the hydrophobic regions of the GFP stick to the HIC beads. Other proteins which are less hydrophobic (or more hydrophilic) pass right through the column. This single procedure allows the purification of GFP from a complex mixture of bacterial proteins. Loading the GFP supernatant onto the chromatography column When students load the GFP supernatant onto their columns, it is very important that they do not disturb the upper surface of the column bed when performing the chromatography procedure. The column matrix should have a relatively flat upper surface. A slightly uneven column bed will not drastically affect the procedure. However, subsequent steps of loading, washing, and eluting should minimize disrupting the column such that beads "fluff up" into the buffer. When loading the GFP supernatant onto the column, the pipette tip should be inserted into the column and should rest against the side of the column...
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...organisms. Nearly every cell in a person’s body has the same DNA. Ribonucleic acid or RNA, is one of the three major macromolecules (along with DNA and proteins) that are essential for all known forms of life. A pipette (also called a pipet, pipettor or chemical dropper) is a laboratory instrument used to transport a measured volume of liquid. Pipettes come in several designs for various purposes with differing levels of accuracy and precision, from single piece glass pipettes to more complex adjustable or electronic pipettes. Lab dish washing Cleaning laboratory glassware isn't as simple as washing the dishes. Here's how to wash your glassware so that you won't ruin your chemical solution or laboratory experiment. You can rinse the glassware with the proper solvent, then finish up with a couple of rinses with distilled water, followed by final rinses with deionized water Water Soluble Solutions (e.g., sodium chloride or sucrose solutions) Rinse 3-4 times with deionized water then put the glassware away. Water Insoluble Solutions (e.g., solutions in hexane or chloroform) Rinse 2-3 times with ethanol or acetone, rinse 3-4 times with deionized water, then put the glassware away. In some situations other solvents need to be used for the initial rinse. Lab chemical stock maintenance • Remove items from the written inventory as they are disposed or used • Record all new orders • Record the received...
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...Types of Chromatography Adsorption Chromatography Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts for the separation of different solutes. Partition Chromatography This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid. Ion Exchange Chromatography In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Molecular Exclusion Chromatography Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones. Affinity Chromatography This is the most selective type of chromatography employed...
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...of their important structural characteristics. This experiment will consist of two parts. The first of which will divide the yeast cells into three of its major macromolecular components: nucleic acids, proteins and polysaccharides. These components are large macromolecules that are quite unique in their composition, structure and function. However, they share a common feature as each macromolecule is composed of repeating subunits, characteristic of the macromolecule. The subunits are linked together by a bond between two adjacent subunits, formed by the loss of water (condensation). Thus, macromolecules can be broken down by the addition of water across the bond, in a process known as hydrolysis. This process was used in the experimental procedure to allow analysis of each individual macromolecule in its subunit form. Proteins are hydrolyzed into amino acids, nucleic acids are hydrolyzed into sugar, base and phosphate, and polysaccharides are broken down into simple sugars. In the second part of the experiment, the principle method of chromatography was used to analyze the macromolecules isolated in part one of the experiment. With this technique, individual molecular species were separated from one another. This separation technique was performed for the protein and nucleic acid components. In addition, for each case, a series of knowns including one unknown...
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...Identification of Unknown Amino Acid Elizabeth Amundson Lab Partner: Mary Witucki Introduction: The goal of this lab was to determine which two amino acids where contained within an unknown mixture by comparing the results of a primary amine test, an amide test, a benzene ring test, a thiol test, and paper chromatography to that of amino acids with known compositions. I hypothesize that alanine and lysine will test positive for primary amines because they are the only amino acids being tested in this reaction that contain a primary amine (-NH2). I hypothesize that glutamine will test positive for amides because it is the only amino acid being tested in this reaction that has an amide, or (R1-N-R2). I hypothesize that tryptophan will test positive for benzene rings because it is the only amino acid being tested that contains a benzene ring. I hypothesize that cysteine will test positive for thiols because it is the only amino acid being tested with a thiol, or (R-S-H). I also hypothesize that the hydrophilic amino acids will have smaller Rf values than the hydrophobic amino acids because the chromatography paper is more hydrophilic than the solvent being used, so they will have a higher affinity for the paper and will not travel very far with the solvent. It is important to be able to identify unknown amino acids so that it can be determined which amino acids are found in cells and proteins. Methods: Paper Chromatography 1. Pick either unknown A, B, or C. 2. Draw...
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...Introduction: Photosynthesis occurs when proteins in plant cells absorb light. Chlorophyll, the most commonly known pigment in plants, absorbs red and blue light, while reflecting the color green. Because of this, many plants are found to be the color green. There are, however, a wide variety of plants that range in color from red to violet. This is due to the different number of plant pigments that compose a plant. One technique that separates and identifies these different pigments is paper chromatography. In paper chromatography, solvent moves up the paper, carrying with it plant pigments. Because plant pigments are not equally soluble in the solvent, they are carried along at different rates, taking up different parts of the visible light system. An interesting question to explore is what other pigments are found in plants besides the obvious, chlorophyll. Using paper chromatography, pigmentation for plant leaves can be determined. Chromatography works because some pigments have a higher affinity for the solvent than others and move at different rates up the filter paper, causing several color bands to be displayed if there is more than one pigment present in the leaves. Based on the bands formed on the filter paper, the retention factor, or Rf , value can be thereby calculated for each pigment. This is done by dividing the distance the pigment traveled by the distance the solvent traveled. In essence paper chromatography will be used to determine what other pigments...
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...Plant Pigments & Absorption Lab SBI4U Metabolic Processes Lab 1 Kevin Salwach P.1 Rm. 208 De Cat November 5 2013 Lab 1 Pigments & Absorption Introduction Autotrophs, a group of organisms to which plants belong, obtain their own food and energy through ways other than hunting and ingestion. Almost all plants use photosynthesis to obtain their energy. Using the CO2 in the air that surrounds them, plants can create the energy needed to survive. The process is split into two parts; the “light” reactions occur by using light from the sun to activate electrons in the photosystem to produce adenosine triphosphate (ATP) the main requirement for metabolism. After this has occurred, the “dark” reactions occur where ATP is reduced to coenzymes in several complex cycles, and with the help of CO2, glucose is produced. This is a simplified explanation of course, as the entire photosynthetic process contains many different reactions and cycles. Overall, this is a very effective way of obtaining energy, and allows a whole manner of organisms to thrive almost anywhere on Earth. Purpose The purpose of this lab is conduct three experiments that answer the questions: - Where to plants conduct photosynthesis? - What photosynthetic pigments do they use? - What wavelength of light works best for photosynthesis? Materials -Microscope -Elodea -Microscope slide / cover slip -Spinach leaves -Mortar / pestle -Distilled water -80% acetone -Sand -Filter paper -Coleus...
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...Microorganisms are referred as predominant source of commercial enzyme (Wiwapat et al., 2002, Kvesitadze, Kvesitadze 1990, Kutateladze et al., 2009). Due to diverse spectrum of applications enzyme production now became a multi-billion dollar business (Bhat, 2000). The market of the technical enzymes showed great diversity both in terms of the applications as well as the consumption. It has been evaluated that global sale of enzymes was $1.7–2 billion in 2005 which is expected to grow in the forthcoming years exponentially with the increase of industrialization. Typically, pectinases shares a huge market value up to $75 million (Godfrey and West, 1996). Pectinases is one of the hydrolytic enzymes that have diverse degree of applications in biotechnological industry. Pectinase is one of the 20 enzymes that have been globally marketed. Pectinase catalyzing the degradation of pectin containing compounds are of huge industrial significance (Spanga et al., 1995). Pectinases are now considered to be central part of complex processing of juice in food industry and removal of sizing agents in textile industries (Kashyap et al., 2001). They cause a drastic increase in filtration efficacy of juices and increase yield by aiding in clarification. (Joslyn et al., 1952) (Brawman 1981). They are also used as wood preservative (Fogarty 1973) and in liquefaction, maceration and extraction of vegetable tissues (Charley 1969 and Bohdziewiez and Bodzek 1994). Different extraction procedures like...
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...Bio Lab Practical #1 Solute- dissolved in solvent Solvent- a substance in which another substance is dissolved Solution- a liquid mixture in which the solute and solvent are uniformly distribute Molecular weight- average mass of a molecule Mole- a measurement of quantity or numbers pH- the measurement of acidity or basicity in an aqueous solution Prokaryotic- single-celled organism that lacks a membrane-bound nucleus, mitochondria, or any membrane-bound organelles Eukaryotic- has true nucleus, nuclear pores, and organelles Organelles- a specialized part of a cell having specific fucntions, cell organ Diffusion- means of passive transport, three types: facilitated, simple, and channel Osmosis- the movement of water from high potential to low potential Brownian movement- random movement of microscopic particles in liquid Dialysis- the separation of particles in a liquid on the basis of differences in their ability to pass through a membrane Dialysis tubing- semi-permeable membrane tubing Biologically Important Molecules: Carbohydrates, proteins, lipids, and nucleic acids – most organic compounds in living organisms Dehydration- removal of water molecule and covalently bonding two subunits Hydrolysis- breaking bonds in subunits by adding water Positive control- contains the variable for which you are test. It reacts positively and demonstrates the test’s ability to detect what you expect Negative control- does not contain...
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...Introduction: In this lab a series of experiments were done to gain a greater understanding of photosynthesis, plant pigments and light absorption and transmittance by/through plant chlorophyll and accessory pigments. Photosynthesis is the process by which plants use energy from light (i.e. the sun, photons, electromagenetic energy), water, and carbon dioxide to produce ‘food’ for themselves, or some for of sugar, like glucose. Photosynthesis has two stages which take place within the chloroplasts of a plant cell, where the photosynthetic pigments, chlorophyll reside. The first stage occurs in the thylakoid of the chloroplasts and is called ‘the light reactions’. During this stage water is split, the oxygen changes (or is evolved) and energy is harvested through photophosphorylation. Photophosphorylation transforms the light energy into the chemical energy of ATP and NADPH, which aid in the next stage, the Calvin Cycle or light independent reactions. During the Calvin Cycle, carbon dioxide is reduced to glucose and oxygen is released as a by product, at which time the ADP and the NADP+ are returned to the light reactions to start the cycle over again, or regenerate. A simplified equation for photosynthesis is as follows: 6CO2 + 6H20 + Light Energy → C6H12O6 (glucose) + 6O2 Photosynthesis is basically a plants version of cellular respiration and is essential for their survival. During the first stage, light energy or electromagnetic energy/radiation...
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...Variable Independent Variable: One or more factors that the scientist varies during the experiment. Dependent Variable: A feature that the scientist measures in order to determine if it changed in response to the independent variable. What solutions were used to test for the 4 types of organic molecules? Iodine- Polysaccharide Benedict’s Reagent- Sugar Biuret Test- Protein Brown Paper Test- Lipids Vegetable Oil- Solubility of Lipids What does a positive test look like? -Iodine test for polysaccharide: dark purple/black/blue -Vegetable Oil test for solubility of lipids: 1 layer -Biuret test for protein: violet color -Benedict’s Reagent for sugar: very high concentration/orange-red How do you convert Celsius to Fahrenheit and vise versa? Degrees Fahrenheit= 9/5 degreesC + 32 degrees Degrees Celsius= 5/9(degreesF - 32 degrees) What is the compound scope magnification equation? eyepiece mag x objective mag What...
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...Red de Revistas Científicas de América Latina, el Caribe, España y Portugal Sistema de Información Científica Duangporn Kantachote, Pakorn Prachyakij, Wilawan Charernjiratrakul, Metta Ongsakul, Yodsawee Duangjitcharoen, Chaiyavat Chaiyasut, Teruhiko Nitoda, Hiroshi Kanzaki Characterization of the antiyeast compound and probiotic properties of a starter Lactobacillus plantarum DW3 for possible use in fermented plant beverages Electronic Journal of Biotechnology, vol. 13, núm. 5, 2010, pp. 1-15, Pontificia Universidad Católica de Valparaíso Chile Available in: http://www.redalyc.org/articulo.oa?id=173318799002 Electronic Journal of Biotechnology, ISSN (Electronic Version): 0717-3458 edbiotec@ucv.cl Pontificia Universidad Católica de Valparaíso Chile How to cite Complete issue More information about this article Journal's homepage www.redalyc.org Non-Profit Academic Project, developed under the Open Acces Initiative Electronic Journal of Biotechnology ISSN: 0717-3458 http://www.ejbiotechnology.info DOI: 10.2225/vol13-issue5-fulltext-1 Characterization of the antiyeast compound and probiotic properties of a starter Lactobacillus plantarum DW3 for possible use in fermented plant beverages Duangporn Kantachote1 1 · Pakorn Prachyakij1 · Wilawan Charernjiratrakul1 Metta Ongsakul · Yodsawee Duangjitcharoen2 · Chaiyavat Chaiyasut2 Teruhiko Nitoda3 · Hiroshi Kanzaki3 1 Department of Microbiology, Faculty of Science, Prince of Songkla University,...
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...it be due to the differences in pH?” ( ). This topic was important to Lauren, because while in the doctor’s office she read an article that discussed a lawsuit pertaining to California’s Proposition 65. The magazine article discussed the hidden danger of carcinogens in the food we eat. Lauren was surprised to find that more people had no idea about the dangers of carcinogens in our food. Lauren collected data for analyzation in three different stages. In the first stage Lauren marinated all of the chicken at home in five different marinades. The second stage was completed at Penn State University and she extracted the chemicals, changed the pH, and ran it through the lab equipment which in turn separated the compounds. In the third and final stage Lauren ran the samples through a high pressure liquid chromatography mass spectrometer, which separated the compounds and analyzed the chemicals, and told her exactly how much carcinogens she had in the chicken. Lauren’s research came out with the following results; marinating chicken in lemon juice decreased the carcinogens by about ninety-eight percent. Salt water and brown sugar marinades also worked to decrease the carcinogens by about sixty percent. Olive oil only slightly decreased the carcinogens and soy sauce results turned out to be inconclusive. A health services administrator could possibly use...
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... . . . . . . . . . . . . . . 2.4.2 Diet . . . . . . . . . . . . . . . . . . . . . . . 2.4.3 Using Drugs to Reduce Detection Times . . . 3 Test Methods 3.1 Substances that are Detectable . . . . . . . . . . . 3.2 DrugAlert . . . . . . . . . . . . . . . . . . . . . . . 3.3 Gas Chromatography . . . . . . . . . . . . . . . . . 3.4 Gas Chromatography / Mass Spectrometry . . . . . 3.5 Hair testing . . . . . . . . . . . . . . . . . . . . . . 3.6 High Performance Liquid Chromatography . . . . . 3.7 ImmunoAssay . . . . . . . . . . . . . . . . . . . . . 3.7.1 Radio ImmunoAssay (aka AbuScreen) . . . 3.7.2 Enzyme Multiplied Immunoassay Technique 3.7.3 Fluorescence Polarization ImmunoAssay . . 3.8 PharmChek . . . . . . . . . . . . . . . . . . . . . . 3.9 TestCup . . . . . . . . . . . . . . . . . . . . . . . . 3 11 13 13 13 15 16 16 16 17 17 18 18 18 19 19 21 21 21 22 24 24 25 25 25 26 26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 CONTENTS 3.10 Thin Layer Chromatography . . . . . . . . . . . . . . . . . . . 26 4 Test...
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...AP Biology Exam Review: Lab Essays At least one essay (FRQ) on the exam will be based on an AP laboratory. To prepare for this question, review the objectives for all twelve laboratory exercises. The College Board does not necessarily expect that you have completed that lab, but rather that you have investigated the objectives of the lab. You may be asked to “design an experiment to determine….” You don’t necessarily need to create a new lab; if you have done an activity that would answer the question, simply describe it. For a good response, you should include the following. 1. State a hypothesis [as an “if…..(conditions), then….(results)” statement] Be sure your hypothesis is testable. 2. Identify the variable factor. 3. Identify the control. Be certain to explain the control for the experiment. 4. Hold all other variables constant. 5. Manipulate the variable. 6. State how you would measure the results. 7. Discuss the expected results. Relate the results to your hypothesis. 8. Include steps to replicate or verify. You may be asked to graph data. Be sure to use a graph that is appropriate for you data. Bar graphs are used when data points are discrete (not related to one another), while line graphs are used with the data are continuous. If there is a data point at zero, be certain to extend your line to 0, but do not extend the line to 0 if there is no data point at zero. Other points to keep in mind: ...
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