The Crucial Concentration

Transcription

1 The Crucial Concentration Investigating Unknown Quantities of Protein Using the Lowry Assay Maryland Loaner Lab Teacher Packet Version 27June17_mks

2 Table of Contents T E A C H E R M A T E R I A L S Materials and Supplies 3 Correlation to Standards 6 Introduction and Overview for Teachers 10 Background Information for Teachers 12 Facilitation Guide 14 Pre-Laboratory Information for Teachers 19 Laboratory Preparation for Teachers 25 Teaching the Lab Activity 30 Answer Keys to Student Worksheets 33 S T U D E N T M A T E R I A L S Nutrition Facts Scavenger Hunt S-1 Macromolecules Scavenger Hunt S-2 Roy, Gee, and Biv s Micropipette Challenge S-4 Lab Background for Students S-5 Lab Procedure for Students S-6 Data Table S-11 Graph S-12 Summary Questions S-13 C-E-R Chart Report It! Colorimeter Demonstration Worksheet S-14 S-15 S-16 2 P a g e

3 Materials and Supplies The following materials are provided by MDLL: The following amounts are for 1 class set. Material Number Comments Return Instructions Teacher Binder 1 Contains all info necessary for completing lab Yes Bag labeled Materials for Pre- Lab Activity Micropipette Challenge - 10 (15 ml) conicals of Red water for Micropipette Challenge -10 (15 ml) conicals of Yellow water for Micropipette Challenge - 10 (15 ml) conicals of Blue water for Micropipette Challenge For Pre-Lab Activity- Micropipette Challenge. Each group gets a red, yellow and blue tube. Yes. Return unused portions. Bag labeled Solution 1and Solution 2 for Lab Activity Bag of empty tubes labeled Cu Reagent Bag containing 10 empty 15ml conicals labeled Folin- Phenol for Folin- Phenol Reagent Bag of tubes of empty distilled water labeled dh2o tubes - An empty bottle labeled Solution 1 (110 ml). Teacher will make Solution 1 using instructions on page 15-1 microcentrifuge tube per class set labeled and filled with Solution 2 (220 ul) -1 flat bottom tube per class set labeled Na 2 CO 3 (sodium carbonate) 10 empty (15ml) conical tubes labeled Cu Reagent 10 (15ml) empty conical tubes labeled Folin-Phenol 10 (15ml) conical tubes labeled dh2o Sol 2 to be added to Sol 1 by teacher, see prep directions on page 15. Keep at room temperature. See Laboratory Preparation for Teachers for details on using Na 2 CO 3 (sodium carbonate) Teacher will prep Cu Reagent (Solution 3) and transfer 10 ml into each conical tube. 1 per group. MUST BE MIXED DAY OF LAB. Keep at room temperature. - Rinse and return Solution 1 bottle - Dispose of Solution 2 microcentrifuge tube -Return empty and unused flat bottom tubes of Na 2 CO 3 (sodium carbonate) Clean and return 1 conical tube per group If used, do not return 1 tube of distilled water for each of 10 groups Yes, empty tubes rinsed and dried 3 P a g e

4 Materials and Supplies Insulated Bag(s)* containing: Unknown Sports Drink A Unknown Sports Drink B Unknown Sports Drink C Stock Protein *number of bags depend on class sets ordered Bag labeled Teacher Set of Standards for Colorimeter Demo - 10 (15ml) conical tubes of unknown sports drink A - 10 (15ml) conical tubes of unknown sports drink B - 10 (15ml) conical tubes of unknown sports drink C - 10 (15 ml) conical tubes labeled Stock Protein (0.5ug/ul) - Teacher Demo Set (8 (50 ml) conicals) Each of 10 groups receives: - 1 (15ml) tube of Unknown Sports Drink A - 1 (15ml) tube of Unknown Sports Drink B - 1 (15ml) tube of Unknown Sports Drink C - 1 (15ml) tube Stock Protein (0.5ug/ul) MUST BE KEPT IN THE REFRIGERATOR - Set of 8 (50 ml) conicals of blue water of known concentration (6) and conicals of blue water of unknown concentration (2) Yes, empty tubes rinsed and dried Return inside insulated bag Return Demo Set filled (do NOT empty) µl 10 1/group Yes micropipettes 1000 µl tips 5 boxes 1 box per 2 groups (tips are Yes, unused tips blue) 200 µl micropipettes 10 1/group Yes 200 µl tips 5 boxes 1 box per 2 groups (tips are Yes, unused tips yellow) Teaspoons 10 1/group Rinse, dry, and return Insulated Bag & 1 Return freezer pack Tupperware container labeled Glass test tubes ~170 test tubes 6/group (Micropipette Challenge) 8/group (Lab Activity) Return UNUSED glass test tubes and container Sharpies 10 For students to label test Yes tubes Cuvette sets 10 sets of 8 cuvettes each, labeled 1-5, A, B, C 8 per group to use in colorimeter Yes, empty tubes rinsed and dried Cuvette racks 10 1 per group to hold Yes cuvettes Test Tube Rack 10 1 per group Yes Disinfectant Wipes 1 bottle Please wipe down Yes pipettes Colorimeter and LabQuest Data Unit 3 Each kit will have 3 kits to be shared among student groups Yes 4 P a g e

5 Materials and Supplies Lab flashlight 1 For teacher colorimeter demo Kimwipes 3-6 Place one by each colorimeter to clean smudges from cuvettes Yes Yes Materials provided by Teacher: Description Quantity Comments 1-2 bag lentils (red preferred, Provide roughly 1/3 c Used with What s in Your Food? but any color lentil or a substitute dry bean will work) dried lentils per lab group 1 4 lb bag of white sugar Provide roughly ½ c sugar Used with What s in Your Food? Beakers or cups (may be disposable) per lab group 4/lab group Use 1 for sugar, 1 for lentils, then 2 empty/lab group for What s in Your Food? Food containers and labels ~2/group Can have students bring in their own food packages and labels. Graduated Cylinder (10ml) 1 Used to measure Cu Reagent Graduated Cylinder (1000 ml) 1 Used to prepare Solution 1 Parafilm Enough to cover beaker Used to prepare Solution 1 to mix solution (optional) Disposable Cups 10 1 per group waste container for lab 2N Folin-Phenol Reagent 10ml of 2N Folin-Phenol for each class set 60 ml Distilled Water per class set For each class set- 10 ml for Folin-Phenol Solution and 50ml for Solution 1 2N Folin-Phenol Reagent can be ordered online at duct/sial/f9252?lang=en&region=us Cost: $47.00 per 100 ml For use by teacher to make Folin-Phenol solution and Solution 1 NaOH (sodium hydroxide) 0.8 g per class set Used to make Solution 1. 1N NaOH can be ordered at P15983C670.aspx?utm_source=google&ut m_medium=shop&utm_campaign=prod Cost: $23.50 for 500 grams Reusable 50ml conical tube or beaker 1 conical tube or beaker Used to prepare Folin-Phenol Reagent SAFETY: The classroom teacher must instruct students with basic laboratory safety rules and provide gloves and goggles for student use with the laboratory activity. 5 P a g e

6 Connections to Standards Next Generation Science Standards Performance Expectations: Students ability to complete the following performance expectation(s) will be supported by participation in this activity. HS-LS1-1: Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins, which carry out the essential function of life through systems of specialized cells. HS-PS1-2: Construct and revise an explanation for outcome of a simple chemical reaction based on the outermost election states of atoms, trends in the periodic table, and knowledge of the patterns of chemical properties. HS-PS1-7: Use mathematical representations to support the claim that atoms, and therefore mass, are conserved during a chemical reaction. Dimension NGSS Code or citation Corresponding student task in activity Disciplinary Core Idea Practice LS1.A: Structure and Function Systems of specialized cells within organisms help them perform the essential functions of life. PS1.B Structures and Properties of Matter The fact that atoms are conserved, together with knowledge of the chemical properties of the elements involved, can be used to describe and predict chemical reactions. Planning and Carrying out Investigations Conduct an investigation and/or evaluate and/or revise the experimental design to produce data to serve as the basis for evidence that meet the goals of the investigation. Construct an explanation In the macromolecules, students will consider and explore the role of macromolecules (including proteins) in the cell and the body. Students will use the results of a chemical reaction (Lowry Assay) to determine the absorbance values of known quantities of protein. They will then use Beer s law to calculate the concentration of sports drinks for which they have absorbance values. Students will conduct an investigation to determine which of three sports drinks has the most protein. Students will make a claim as to which sports drink contains the most protein and use data from their 6 P a g e

7 Connections to Standards Construct an explanation using models or representations. investigation as evidence to support their claim. They will provide scientific reasoning to connect their Construct a scientific explanation based on valid and reliable evidence obtained from sources (including students own experiments) and the assumption that theories and laws that describe the natural world operate today as they did in the past and will continue to do so in the future evidence to their claims. Crosscutting Patterns Students will observe the patterns of color change in Concept Use mathematical representations to identify certain patterns. their samples after performing the Lowry Assay. Students will plot their data on a graph, and use Beer s Law to determine concentration of unknown proteins. Cause and Effect Students will explore how specific chemical reactions Students suggest cause and effect relationships to explain and predict behaviors in complex natural and designed systems. cause color changes based on protein concentration. Students will use empirical evidence based on cause and Students will use cause and effect relationships to predict phenomena in natural or designed systems. effect of specific chemical reactions to make claims about the amount of protein in sports drinks. Energy and Matter Students will use chemical reactions to quantify the Energy cannot be created or destroyed. It only moves between one place and other, pace, between object and/or field, or between systems. concentration of a colorless protein. Nature of Science Scientific Knowledge is Based on Empirical Evidence Science knowledge is based upon logical and conceptual connections between evidence and explanations. Scientific Models, Laws, Mechanisms and Theories Explain Natural Phenomena Theories and laws provide explanations in science, but theories do not with time become laws or facts. 7 P a g e

8 Connections to Standards Laws are statements or descriptions of the relationships among observable phenomena. Scientific Knowledge Assumes an Order and Consistency in Natural Systems Science assumes the universe is a vast single system in which basic laws are consistent. Connections to Common Core State Standards English Language Arts/Literacy Mathematics RST HSF.IF.B.5 RST PRACTICE.MP3 RST PRACTICE. MP4 RST PRACTICE.MP5 RST PRACTICE.MP6 RST W W Advanced Placement Standards: Chemistry Standard Enduring Understanding 1.D. Atoms are so small that they are difficult to study directly; atomic models are constructed to explain experimental data on collections of atoms. Essential Knowledge 1.D.3.c: The amount of light absorbed by a solution can be used to determine the concentration of the absorbing molecules in that solution, via the Beer-Lambert Law. Science Practice 2: The student can use mathematics appropriately. Associated Activity in Activity Students will use perform a Lowry Assay on protein samples of known and unknown concentrations. They will use a colorimeter to measure the amount of light being absorbed in each sample and use Beer-Lambert Law to calculate the concentration of protein in the unknown samples. Students will use Beer s law to determine the concentration of the unknown sports drinks. 8 P a g e

9 Connections to Standards Science Practice 5: The student can perform data analysis and evaluation of evidence. Science Practice 6: The student can work with scientific explanations and theories. Students will look for patterns and relationships in the data and evaluate that data to answer the question of which sport drink has the highest concentration of protein. Students will construct a scientific argument to support their claims about which sport drink has the highest concentration of protein. The argument will include evidence and scientific reasoning. 9 P a g e

10 Introduction & Overview for Teacher The Crucial Concentration Loaner Lab Module has three parts: A pre-laboratory classroom activity ( What s in Your Food? ) where students explore the importance of protein and carbohydrates in foods, and are introduced to the career of food scientist. Pre-laboratory classroom skills activities ( Roy, Gee, and Biv s Micropipette Challenge and Colorimeter Teacher Demo ). Roy, Gee, and Biv s Micropipette Challenge introduces students to the use of a micropipette. Colorimeter Teacher Demonstration illustrates how the colorimeter works and provides an opportunity for students to practice calculations, graphing, and creating a standard curve. A main laboratory activity ( The Power Drink Challenge ) that allows students to use the Lowry Assay to determine which of three sports drinks has the highest concentration of protein. Scientists are often faced with the challenge of determining the concentration of a substance in a solution. For example, they may need to measure levels of proteins, cholesterol, glucose, or the rate of enzymatic activity. This investigation focuses on a colorimetric assay commonly used to measure protein concentrations called the Lowry Assay. The Lowry Assay requires a series of standard protein solutions to create a standard curve. The standard curve is used to measure the quantity of protein in an unknown solution. Because the protein used to make the standards is colorless, a chemical reaction is required to produce a color. The intensity of that color is in direct proportion to the amount of protein present. The concept of developing standards for measurement is frequently applied to solve quantification problems. A standard is a tool, made up of known increments or units, which is used to measure something. This idea is often quite familiar to students although they may not recognize it as such. A rule or tape measure, for example, serves as a standard when measuring the lengths of objects. Measuring the concentration of a substance in a solution also requires development of a standard. This investigation is organized into two parts pre-laboratory activities ( What s in Your Food?, Roy, Gee, and Biv s Micropipette Challenge, Teacher Colorimeter Demo ) and a main laboratory activity ( The Power Drink Challenge ). In the first pre-laboratory activity, What s in Your Food?, students work through a scavenger hunt to study food labels, then measure out the amounts of carbohydrates and protein in the food using a conversion of grams to teaspoons. A second scavenger hunt, the Macromolecule Scavenger Hunt, challenges students to learn about the roles each of the macromolecules plays in our bodies. These activities help students understand the importance of quantifying nutrient 10 P a g e

11 Introduction & Overview for Teacher concentrations in food, thus setting the stage for the main lab activity, The Power Drink Challenge. The second pre-laboratory activity, Roy, Gee, and Biv s Micropipette Challenge, allows students to practice using micropipettes. Measuring volumes precisely is critical in this lab and this fun challenge offers students ample opportunity to practice their pipetting techniques. For student unfamiliar with the colorimeter, the teacher demonstration using the colorimeter can be completed at any time prior to the students using the colorimeters. A complementing student worksheet encourages more practice creating standard curves or calculating concentrations. Following the pre-laboratory activities, students apply the concepts that they learned to perform the main laboratory activity, The Power Drink Challenge. In this activity, students determine which of three sports drinks contains the highest concentration of protein. This activity concludes with a report supported by summary questions and a Claim-Evidence Reasoning (C-E-R) chart. 11 P a g e

12 Background Information for Teachers Food scientists, biochemists, and others analyze food to determine the concentrations of various macro- and micro- nutrients, such as simple and complex carbohydrates, protein, lipids (fats), vitamins, sodium, etc. One reason for these analyses is to satisfy food labeling regulations managed by the Food and Drug Administration (FDA). The FDA requires labeling of food products for the purpose of informing consumers of the concentrations of nutrients in their foods to promote informed consumption and healthier diets. Other reasons some scientists conduct nutrient analyses are to attempt to mimic and to improve a food for marketing. By knowing the nutrients present and the nutritional targets for those nutrients, these scientists can work with nutritionists and others in the food development industry to improve the health of some marketed or packaged foods. Each chemical analysis process for nutrients is specific to the nutrient under investigation. Some nutrient tests serve as a screening for the nutrient while other tests determine percentages or concentrations of the nutrient. Two carbohydrate screening tests differentiate between simple and complex carbohydrates. Biuret s reagent tests for the presence of simple sugars, such as those found in candies, sweets and soft drinks. The color changes can provide a broad differentiation on simple sugar concentrations. Starches, which are complex carbohydrates produced by plants, are easily identified by exposing the food to iodine, which turns dark blue-black in the presence of starch. By contrast, the Lowry Assay, which is the main laboratory focus of this lab module, measures the amount of protein found in food. The Lowry Assay uses a set of known protein concentrations to build a standard curve that is used to determine the concentration of protein in a sample. The macronutrients are also known as biological macromolecules. In order to help students understand the importance of the macromolecules in their diets and to explain why foods are labeled with nutrient information, students explore the macromolecules with a scavenger hunt. There are four classes of biological macromolecules, carbohydrates, proteins, lipids and nucleic acids. Carbohydrates serve as the primary energy source for cells and are important structural components in cells and the body. Simple carbohydrates burn fast, providing a burst of energy followed by a crash. Complex carbohydrates and proteins sustain energy for a longer period of time and serve other functions in the body, so are generally considered preferred dietary options. Protein is a macromolecule which consists of a string of amino acids. The cell s DNA dictates the order of the amino acids, and the amino acid sequence determines the protein s function. Twenty amino acids combine in different sequences to form all of the proteins in the body. Humans cannot synthesize nine of the twenty; those nine must be included in the diet. The other eleven can be synthesized by humans, if the necessary building blocks are consumed and available. Muscle, enzymes and some hormones consist of protein, and some protein can be also used for energy. 12 P a g e

13 Background Information for Teachers Lipids, or fats, also serve in structural roles, assist the absorption of some vitamins and other nutrients, and also store energy. In food, lipids frequently carry flavor molecules and play large roles in texture. Depending on their chemical composition, lipids can be solid or liquid at room temperature. Nucleic acids, the only macromolecule not also classified as a macronutrient, consist of sequences of five nucleotides (thymine, adenine, guanine, cytosine, and uracil). The first four nucleotides combine in a double stranded helix to form DNA; the last four combine in single strands to form RNA. DNA encodes the genes and genome for the organism. RNA has several roles. For example, RNA serves as a template to make proteins and carries the correct amino acid to build the primary sequence in protein. The main laboratory focus of this lab module is the Lowry Assay, which quantifies protein concentration and uses Beer s Law. After exploring proteins as one of the macromolecules, students use the Lowry Assay to create a standard curve and analyze three different protein drinks for the concentration of protein present in each. In the Lowry Assay, chemicals bind to colorless protein so it can be measured using a colorimeter. Copper reagent (Cu Reagent), the first reagent in the assay, binds to protein. The complex formed is still colorless until a second reagent, Folin-Phenol, binds to the copper-protein complex and forms a blue-gray color. The deeper the blue-gray, the greater the protein concentration. Colorimeters read the absorbance, and the unknown protein drinks protein concentrations can then be compared to a standard curve which is also created by the students. Teachers could chose to extend this lab by analyzing some food for carbohydrates or other nutrients, or exploring other aspects of food and nutrition. 13 P a g e

14 Facilitation Guide Pre-Laboratory Engagement & Exploration What s in Your Food activity (30-45 minutes) 1. To engage students, show food products they might eat or ask them to pull out the food examples they brought in with them. Ask the students why we eat. Accept all answers. Eventually, but not necessarily at this point, students will come to recognize that they eat for energy and to gain nutrients, so their food must contain those nutrients. 2. Organize students into 10 lab groups. Explain that the students will participate in a scavenger hunt using food items. Distribute the Nutrition Facts Scavenger Hunt worksheet in Student Sheets and ask students to select two food products to use on the hunt, then have students complete the Nutrient Facts Scavenger Hunt. 3. Explain that 4 teaspoons equals roughly 1 gram. Distribute the sugar and one empty cup. Ask groups to select one food and measure out the amount of carbohydrates found in that food using the sugar. Ask groups to hold up the amount of sugar in their cups and the food products. Ask, Were you surprised by the amount of sugar present? Why or why not? 4. Distribute the lentils or beans and a second empty cup. Ask students to measure the protein using lentils and the conversion of 4 teaspoons equals 1 gram. Again, ask students to share their results, to whether they were surprised. 5. Ask students to compare the amount of carbohydrate to protein in their foods, and make determinations on what is healthier. Ask how they are making these determinations of health. Facilitate a brief discussion about the roles of carbohydrates and proteins in our bodies. 6. To continue the scavenger hunt theme, have students work in groups to complete a scavenger hunt on the four macromolecules, three of which are macronutrients. Distribute the Macromolecule Scavenger Hunt Student Sheets and provide access to the internet, text and book materials, etc. Also consider rules to support other learning goals. For example, require students to document their source and/or require a minimum number of sources. A time limit, perhaps 10 minutes depending on time available and resource accessibility, can be set. 7. After the scavenger hunt, revisit the question of healthy food asked earlier. Review and reiterate with students that while carbohydrates and proteins are really important to include in a diet, protein tends to be of great interest for muscle growth and for long- 14 P a g e

15 Facilitation Guide term energy release, and so are highly valued in power drinks or products marketed for workouts and exercise recovery. 8. Ask how we get the information that is provided in the food labels. Introduce the career field of food scientists. This video link ( provides a good overview of the career field. 9. Show the students the three protein drink advertisements and ask what they notice about the advertisements (all three claim to have the highest concentrations of protein in any sport drink). Explain that the students will be acting as food scientists by testing three protein drinks for the protein concentration using the Lowry Assay. But, first, there are some laboratory skills necessary in order to be successful in the assay. Pre-Laboratory Exploration and Lab Skills Roy, Gee, and Biv s Micropipette Challenge (30-45 minutes) Colorimeter Teacher Demonstration (10-30 minutes) 10. Introduce the students to the parts of the micropipette and explain how to use them. 11. Ask students to complete the steps of the challenge. As students complete the challenge, review the volume in all six test tubes for each group. Each tube should have the same volume (440 µl). If volumes are not equal, work with the students to make sure they can accurately micropipette. The ability to micropipette is essential in successfully completing the Lowry Assay. 12. If students are unfamiliar with colorimeter, allow minutes to demonstrate how the colorimeter works. Directions for a teacher demonstration are included in the Teaching the Laboratory Activity of this manual and materials are included in the kit. A student sheet (S-16) complements this teacher demonstration if the students need practice calculating concentrations, graphing data points, or creating a standard curve. (NOTE: This step can be completed at any time before students use the colorimeters in their main laboratory procedure.) Main Laboratory Exploration (45 minutes) Lowry s Assay to determine protein concentration of three drinks (45 minutes) Below is an outline of the main laboratory activity. Detailed instructions and teacher support notes are available under the Teaching the Laboratory Activity section of this manual. 15 P a g e

16 Facilitation Guide 13. Distribute the student worksheet, Lab Background (S-5 S-6) to students and ask them to read and work through the background information. This can be done in partners or individually, then shared with the class. Reiterate the flow of the Lowry Assay, so that they understand the purpose of each of the reagents. 14. Remind students that they are acting as food scientists to determine the amount of protein in three sports drinks. Point out that the drinks are not identified so that the test is blind and ask students why that might be important (to reduce bias). 15. Ask students how they will be able to determine the amount of protein in each drink (the darker the blue-gray, the more protein). Introduce Beer s Law. 16. Lead students along a brief discussion so they recognize that they must also use controls, and that the controls they use will be used to construct a standard curve. Distribute the Lab Procedure (S-7 - S-10) and Data Table (S-11). 17. Review lab safety, then have students complete the steps of the procedure. Remind students to complete the Data Table (S-11) as they collect their data, and to complete the necessary calculations. Post-Lab Explanation Graphing and Analysis of Results (15-30 minutes) Summary Questions, C-E-R & Report(varies) 18. Distribute graph paper (S-12) to students, or ask students to enter data into a computer graphing program. Have student use a best-fit line for the standards. 19. Plot the three data points for Unknown Sports Drinks A, B, and C. Then use the graph to determine the concentration of the protein in each drink. Survey the class to see which drink actually had the most protein. 20. If the class disagrees on the drink with the most protein, lead a discussion on what a real lab would do in this situation and factors that might influence different results. 21. The post lab materials are organized so students first complete Summary Questions (S- 13), then a Claim-Evidence-Reasoning (C-E-R) chart (S-14), and they use these two components to assist as they produce a report (S-15). a. The Summary Questions check understanding of the flow of the lab, the purpose of steps in the procedure, and guide interpretation of their results analysis. 16 P a g e

17 Facilitation Guide b. The C-E-R helps students to organize and construct a scientific explanation. Students generally understand how to make a claim (answering the question posed), and are usually readily able to apply concepts in lab and background information to provide evidence for their claim. However, students may struggle to adequately provide the reasoning why the evidence fits the claim. c. The report asks students to include certain information but leaves it up to the students to decide how to present the requested information. Students may benefit from working in pairs to collaborate on this, much like scientists generally co-author articles with their lab teams. Extension Activities (time varies) Analyzing food for other nutrients Exploring wave length Elaborate on the Beer-Lambert Law Career Exploration Below is a list of ideas for extension activities. 1. Use the wavelength setting of 635 nm for the colorimeter to lead into a discussion about the electromagnetic and visible spectrums. Include a discussion about light absorption, reflection, and transmission, and why we see the colors that we do. Discuss why the colorimeter is set at 635 nm (which is at the red end of the visible spectrum) and why we see the Lowry Assay results in the tubes as a blue-gray color. (The proteins in the samples absorb light in the red end of the visible spectrum and what gets reflected to our eyes is light in the blue end of the visible spectrum, which is why the colorimeter is set at 635 nm.) 2. Incorporate into the post-laboratory activity an in-depth discussion about the Beer- Lambert Law. Discuss why scientists generally prefer to express the Beer-Lambert Law using absorbance rather than % transmittance (%T). (It s based on the linear relationship that exists between concentration and absorbance, except at very high concentrations.) Have the students take the short Beer s Law quiz available at the above website. 3. Have students design an experiment to test the amount of protein in different foods. For example, the protein content of different cereals could be compared. This requires students to ask their own question and alter the procedures accordingly. 17 P a g e

18 Facilitation Guide 4. Have students conduct food analyses for other macromolecules, such as carbohydrates and lipids. Some online resources, such as McMush (readily available on several websites accessible with a Google search for McMush ), provide protocols and sometimes lesson plans. 5. Have students research careers like food scientists and biochemists, which relate to this lab. 18 P a g e

19 Pre-Laboratory Information for Teachers Pre-Laboratory What s in Your Food? The purpose of this pre-laboratory activity is to engage students in the laboratory investigation by connecting Lowry s Assay to a real-world issue and careers. This activity allows the option to connect to biochemistry and nutrition. The objectives of What s in Your Food? are: Identify and compare the amounts of protein and carbohydrates in foods students eat Explain the dietary sources of macromolecules Understand the roles of macromolecules in the body and in cells Pre-laboratory What s in Your Food? Materials: Per group: ½ cup of table sugar 1/3 cup of lentils Two plastic cups or small beakers Teaspoon 2 food labels or food products Nutrient Fact Sheet Scavenger Hunt (S-1) Macromolecule Scavenger Hunt (S-2 S-3) Notes: Follow the facilitation guide to work students through this activity. This activity is intended as to introduce the concepts, engage students, and allow students to explore macromolecules and the connections to diet and chemical analysis. Consider using a Think-Pair-Share technique to encourage student participation and increase engagement. Sugar and lentils can be reused between classes if they are not mixed in the same cup when measuring them out. Macromolecule Scavenger Hunt questions can be tailored to suit the course objectives. Pre-Laboratory Roy, Gee, and Biv s Micropipette Challenge Roy, Gee, and Biv s Micropipette Challenge allows students to practice using micropipettes. This activity may be performed the day of the main laboratory activity or any time in advance. The goal of this lab is for students to use proper pipetting technique to move around different amounts of colored water. In the end, they should end up with six test tubes with the same amount of liquid in each. The colors will form a rainbow - hence the name, Roy, Gee, and Biv s 19 P a g e

20 Pre-Laboratory Information for Teachers Micropipette Challenge. Roy G. Biv stands for red, orange, yellow, green, blue, indigo, and violet, which are the colors in a rainbow spectrum. By filling out the chart as they go, they can keep track of where they added liquid and where they removed liquid. This provides an important reference for checking for errors if they do not end up with the same amount of liquid in each test tube at the end. They then have an opportunity to practicing converting their units from µl to ml. All students must be reminded about the proper usage of micropipettes to prevent damage to the equipment and also to provide students with accurate results during the main laboratory activity. Be sure that everyone understands how to operate the micropipettes. It is worthwhile to check each student for correct technique before beginning the main laboratory activity. Please remind students that TRYING TO TURN THE PIPETTES PAST THEIR MAXIMUM VOLUME WILL CAUSE THEM TO BREAK. Pre-laboratory Roy, Gee, and Biv s Micropipette Challenge Materials: Per group: 1 test tube rack 6 test tubes Sharpie 1 conical of Blue water 1 conical of Red water 1 conical of Yellow water µl micropipette µl micropipette Blue tip box with tips Yellow tip box with tips Waste container Student Worksheet Roy, Gee, and Biv s Micropipette Challenge (S-4) Instructions for Using the Micropipettes Micropipettes are precision instruments designed to measure and transfer small volumes. They are expensive and must be used with care. Their accuracy is dependent upon their proper use. Different brands of micropipettes vary in the range of volumes they will measure, the type of tips they fit, and the type of device used to set the volume. Setting the Volume All micropipettes have a volume control dial. Determine whether the volume window on your pipette shows tenths of microliters (0.1 µl) or whole microliters in the smallest place, so that 20 P a g e

21 Pre-Laboratory Information for Teachers you can read the scale correctly (it varies with different brands of micropipettes). Each set of micropipettes comes with a laminated card with specific instructions for setting their volumes. Drawing Up and Expelling Liquid Micropipettes have two stops as you depress the plunger to expel liquid. The first stop corresponds to the volume set in the window. The second stop gives a little puff of air to blow out any remaining liquid upon delivery. To draw liquid into the pipette tip, depress the plunger control only to the first stop. If you go to the second stop, you will draw too much liquid into the tip. The most common pipetting error is to go past the first stop, to the second stop, for drawing liquid into the tip (which gives an inaccurate volume). You go to the second stop only when you are letting the liquid out of the tip. Using the Micropipette 1. Select the pipette that includes the volume range you will need. 2. Adjust the pipette to the desired volume by turning the dial. DO NOT turn beyond the volume range for the pipette. 3. Press a new tip onto the pipette firmly (gently tap the pipette into a tip while the tip is in the box). Get a tip without touching it with your hands - this is to prevent contamination of the samples. 4. To draw liquid into the micropipette tip: Depress the plunger to the first stop to measure the desired volume and hold it in that position. Holding the pipette vertically, immerse the tip 1-3 mm into the liquid to be transferred. Draw the fluid into the tip by slowly releasing the plunger. Wait 1-2 seconds to be sure that the full volume of sample is drawn into the tip. If you see air bubbles, there is a problem with your volume and you will need to repeat this step to get the correct volume (either your tip wasn t immersed far enough down into the liquid or you perhaps raised your arm while releasing the plunger). 5. To dispense the liquid: Place the tip into the container where the liquid is to be released, near the bottom. Slowly depress the plunger to the second stop to blow out all of the liquid in the tip. Be careful not to suck liquid back into the tip by releasing the plunger while the tip is in the liquid you just dispensed. 21 P a g e

22 Pre-Laboratory Information for Teachers When done, eject the tip into a waste container by pressing the separate ejector plunger found on the top or side of the micropipette (depending on the brand of micropipette). Golden Rules of Micropipetting 1. Don't rotate the volume adjuster beyond the upper or lower range of the pipette - this can damage it. 2. Don't use a pipette without a tip on it. If this happens, liquid gets into the opening of the pipette and can damage the mechanism inside. 3. Don't lay down a pipette that has a tip filled with liquid. If this happens, liquid can get inside the pipette and can damage it. Notes: Have students follow the directions on the Student Sheet, Roy, Gee, and Biv s Micropipette Challenge, to complete this activity. Remind students to complete the chart as they work through the activity. Be sure to review micropipette use with student groups whose final volumes are not equal. Common micropipette errors include: o Going down to the second stop to draw up liquid o Pushing the tip against the base of the test tube, which can block liquid extraction o Forgetting to change the volume setting o Not securing the tip or overly securing the tip Teacher Demo: How to Use the Colorimeter The purpose of this pre-laboratory demonstration is to Explain and understand how the colorimeter works Provide an opportunity to practice calculating concentrations, graphing data, and creating a standard curve Demonstration Materials: Colorimeter Demonstration Worksheet (S-16 S-17) (optional) Lab flashlight (6) conicals of known concentrations (2) conicals of unknown concentrations 22 P a g e

23 Pre-Laboratory Information for Teachers The Colorimeter Demonstration Teacher Notes The teacher demonstration set contains six conicals of known concentrations and two unknowns (see the table below). Conical Number of drops of blue dye Volume of water added (ml) Concentration (drops/ml) A B C D E F Unknown 1 50 Unknown 2 50 To review concentration with students, ask the students to determine the concentration of drops of blue dye in each conical (S16). Then, to demonstrate how a colorimeter works, shine a flashlight through the conicals so that the light reflects on a white background. Ask the class to assign each color intensity a number from 1 to 10, 1 being the lightest and 10 being the darkest. Record the values assigned by the class for each standard. (This is what a colorimeter does). Next, have the students graph the results with the concentration on the x-axis and the number assigned for color intensity on the y-axis. When the best-fit line is drawn, they will have constructed a standard curve. Demonstrate how the graph can be used to estimate the unknown concentrations of the mystery solutions (based on Beer s Law- see explanation of Beer s Law on page 25). We have purposefully not given the concentration of the unknown solutions in this teacher demonstration. This is because it is important for students to realize that when conducting authentic science, scientists cannot check the back of the book to see if their answers are correct. If your students ask you what the right answer is, use that question as an opportunity to discuss the nature of authentic science. You can ask the students how they think scientists handle this element of uncertainty when they conduct their own research. Ideas that may come up in the discussion include: Scientists may repeat an experiment a number of times to confirm their conclusions are reliable. 23 P a g e

24 Pre-Laboratory Information for Teachers Scientists must carefully record their protocols, procedures and results so they can carefully scrutinize them for consistency. Scientists often share their work and collaborate with others, allowing their methods, results and conclusions to be critiqued and validated. 24 P a g e

25 Laboratory Preparation for Teachers Two reagents in the laboratory exercise, Solution 1, Solution 3 and Folin-Phenol, must be made fresh on the day of use in order to be effective. All protein (Stock Protein and Unknown Protein Samples A, B, and C) arrives aliquoted, but must be refrigerated until use. Solution 1 (110 ml) (Sodium Carbonate/Sodium Hydroxide) Made by teacher To make 110ml of 4% Carbonate in.2 N NaOH (Sodium Hydroxide) (enough for 1 class set), 1. Weigh out 0.8g NaOH (Sodium Hydroxide) and put into a 500ml flask or beaker 2. Add 55ml of distilled water and stir to dissolve. 3. Add 4 g of Na 2 CO 3 (sodium carbonate, provided by MDLL in flat bottom tube) and continue stirring until dissolved. 4. Add 55ml more of distilled water using a graduated cylinder. Place a parafilm over the flask/beaker with solution and invert to mix before adding to a labeled storage bottle. This solution has a long shelf life and can be prepared days ahead of time. Solution 2 (220 µl) (Cupric Sulfate/Sodium Citrate) Provided by MDLL This solution arrives in a microcentrifuge tube marked Solution 2 (220 µl). It may be stored indefinitely at room temperature. This will be used to make Cu Reagent (Solution 3). This amount is for the equivalent of one class set. Solution 3 Copper Reagent Made by teacher 1. Place all prepared Solution 1 (110ml of Sodium Carbonate/Sodium Hydroxide) into the empty bottle labeled Solution Using a 1000 µl micropipette and clean tip, place all of Solution 2 (220µl) to the bottle labeled Solution 1 (110 ml) - be careful to add the entire amount of Solution Cap the bottle and mix very well by inversion. This is now Solution 3, or Cu Reagent. 4. Using a graduated cylinder, aliquot 10 ml of Solution 3 into each of the 10 conical tubes marked Cu Reagent that are provided. There is one tube per group. IMPORTANT: This solution MUST BE PREPARED WITHIN 5 HOURS of being used. Keep at room temperature. 25 P a g e

26 Laboratory Preparation for Teachers Folin-Phenol Made by teacher 10ml of 2N Folin-Phenol Reagent needs to be provided by the teacher for each class set. 1. Place 10 ml of 2N Folin-Phenol Reagent into reusable 50ml conical tube 2. Add 10 ml of distilled water and mix thoroughly 3. Place 2ml aliquots into conical tubes (labeled Folin-Phenol) and keep at room temperature. MDLL will provide these conical tubes. Reuse these tubes for each of your classes, then dispose of tubes after using (do not send back). Stock Protein Provided by MDLL, keep refrigerated until use Stock protein solution (Bovine Serum Albumin) labeled Stock Protein (0.5 µg/µl) has already been aliquoted into 10 conical tubes. Unknown Protein Samples A, B, and C Provided by MDLL, keep refrigerated until use 30 conical tubes have already been filled with 1.5 ml of the samples of unknown protein concentrations (A, B, and C). Unknown A has µg/µl BSA, Unknown B has µg/µl BSA, and Unknown C has µg/µl BSA. IMPORTANT: Keep protein samples refrigerated until ready to use. Do not tell students the concentration of the unknowns! Colorimeter Demo Materials Provided by MDLL Eight conicals with known concentrations and two with unknown concentrations are provided, along with a lab flashlight. These remain sealed and are returned with the kit. Colorimeter and LabQuest Data Collection Unit Provided by MDLL Students will use a Vernier colorimeter, plugged into the handheld LabQuest data collection unit, to determine the absorbance values of their known protein concentration standards (test tubes #1-5) and their 3 unknown sports drink samples (test tubes A, B, and C). Laminated instructions are provided that can be placed next to each colorimeter for students to refer to. Instructions for using the colorimeter are as follows: Fig. 1. LabQuest Data Collection Unit 1. Plug colorimeter into CH 1 on the Vernier LabQuest (Fig. 1). 26 P a g e

27 Laboratory Preparation for Teachers 2. Turn the LabQuest on by pushing the power button in the upper left corner (Fig. 1). 3. Wait 5 minutes for the colorimeter to warm up. 4. Make sure the colorimeter is set to measure at wavelength 635 nm (Fig. 2) - this will be indicated by a lit green light under the label 635 nm. If it is not at the correct setting, hit the < or > button to select 635 nm. 5. Students will have already transferred 2 ml of each of their protein solutions (test tubes 1-5 and A, B, and C) into their cuvettes (labeled #1-5, and A, B, Fig. 2. Colorimeter and C). Please make sure students use the provided cuvette rack to transport their samples to wherever the colorimeter is located (the colorimeters are shared among groups). This will prevent spilling and loss of their samples. If a group does spill or otherwise lose their sample, we recommend that they take absorbance readings on their remaining samples so they understand how the colorimeter works, but it may be best to have them use another group s data when they complete their graph. 6. Each group will need to perform their own calibration. They will do this by putting the sample from their cuvette 1, which contains no protein, into the colorimeter. To do this they will: a. Open the colorimeter lid b. Clean the cuvette with a kimwipe and gently place cuvette 1 into the colorimeter with the clear side pointing to the arrow on the top of the cuvette slot of the colorimeter (Fig. 3). DO NOT force it down; gently insert it only as far as it easily goes. If you force the cuvette too far into the colorimeter, it becomes stuck in the plastic insert that holds the cuvettes and is very difficult to remove. c. Press the CAL (calibrate) button on the colorimeter (Fig. 2) and hold until the red LED begins to flash (this is usually very quick - less than one second). Check to see that the reading on the LabQuest unit is either or (see Fig. 1). You have now calibrated the colorimeter for the rest of that group s cuvettes. Remember, EACH group must recalibrate using their own sample # 1 (which contains no protein). d. Remove cuvette 1. e. Insert cuvette 2. Record the absorbance on the student data sheet. It may take a second or two for the reading to stabilize. Remember, you do NOT want 27 P a g e

28 Laboratory Preparation for Teachers to hit CAL again; you only do that with the first sample. Gently remove the cuvette. f. Repeat above step (step e) for samples 3-5 AND for each unknown sample (A, B, and C). Make sure students record each absorbance reading on their data sheet. g. Please make sure to gently rinse and dry the cuvettes as we do reuse them. Any scratches to the cuvettes will affect future absorbance readings. Please note that while the LabQuest can be programmed to produce an Absorbance vs. Concentration graph (based on Beer s Law), we are purposefully having the students plot their own graph. We have found this is an essential activity for students to fully understand the connection between the standards they made and how they are used to determine the concentration of unknown samples. It also provides an opportunity to reinforce the students graphing skills. Laboratory Materials: Prepare 10 Student Workstations Per station: Copies of the Lab Background (S-5 S-6), Lab Procedure (S-7 S-10), Data Table (S-11) and Graph (S-12) for each student Test tube rack 8 empty test tubes Cuvette rack 8 empty cuvettes Sharpie 1000 µl micropipette One blue box of 1000 µl micropipette tips (1 box/2 student groups) 200 µl micropipette One yellow box of micropipette tips (1 box/2 student groups) One disposable cup (waste container for tips) (provided by teacher) Unknown Samples A, B, and C in conical tubes (2.0 ml each)* Stock Protein (0.5 µg/µl) sample in conical tube (1 ml) * Cu Reagent (Solution 3) in conical tube (10 ml aliquoted by teacher) * Folin-Phenol reagent in conical tube (2 ml) * Distilled water, dh20, in conical tube ( 6 ml) *Make sure all liquids are thoroughly mixed (by inversion) before placing at stations Shared Equipment for Multiple Student Stations LabQuest Data Unit and Colorimeter. A minimum of 3 and a maximum of 6 of both of these units (depending on how many we have available) are provided with each kit. Since the groups will have to share, we recommend having the colorimeters and 28 P a g e

29 Laboratory Preparation for Teachers attached LabQuest units set up in a central location and have the students bring their samples to the colorimeter. Place one of the laminated set of directions next to the colorimeter to assist students (but please also give verbal instructions as well). Place a box of kimwipes next to each unit to clean off the cuvettes before they are placed into the colorimeter. 29 P a g e

30 Teaching the Laboratory Activity The purpose of the main laboratory activity, The Power Drink Challenge, is to use the Lowry Assay to determine which of three sports drinks has the highest concentration of protein. The objectives for the main laboratory activity are: Apply the general concept of quantification to a Lowry Assay Collect data using a colorimeter Graph data to produce a standard curve Using a standard curve, determine the concentration of a protein solution Developing the Concept for the Assay Give each student a copy of the Lab Background Student Worksheets (S-5 S-6). Have them read the background information about The Power Drink Challenge and challenge them to find the concentration of protein in three unknown sports drink samples. Ask each group to develop an idea for solving the problem. Lead a discussion about their ideas and record the highlights on the board. Now show students the three protein solutions: A, B, and C. Students should realize that the protein solutions have no color and, therefore, cannot simply be visually inspected to indicate how much protein is in the solution. Inform students that a chemical reaction can be used to add color. The reaction they will use is a two-step assay called the Lowry Assay, diagrammed in their student worksheets. Once the Lowry Assay has been performed, a colorimeter can be used to measure the absorbency of each of the protein standards as well as the unknown samples. At this point, students will use Beer s Law to determine the concentration of their unknown samples. Beer s Law states that the concentration of a chemical substance is related to the amount of light absorbed by the sample. The equation for Beer s Law is A =ɛɓс where A = absorbance, ɛ is the molar extinction coefficient (L/mol cm), ɓ is the pathlength of the sample cell (cm), and с is the concentration of the substance that absorbs light. Notice that both ɛ and ɓ must be known to directly calculate с from A. If ɛ and ɓ are not known (as in this case), then a standard curve of absorbance vs. concentration, using standards of known concentration, can be constructed. The slope of the line is equal to the product, ɛɓ. The relationship between A and c must be linear to satisfy Beer s Law. In the Lowry assay, you are effectively constructing a standard curve assuming Beer s Law and determining the product, ɛɓ. Main Lab Activity: Create the Protein Standards and Perform the Lowry Assay Each group of students receives a stock protein solution (0.5 µg/µl BSA or Bovine Serum Albumin) and distilled water. They will create a set of standards by making a series of protein 30 P a g e

31 Teaching the Laboratory Activity solutions of known concentrations. The students next perform the Lowry Assay on both the set of protein standards and three unknown protein solutions (labeled A, B, and C). Use the Colorimeter and Record the Data Teachers must provide instructions to the students for using the colorimeters. Please also place a laminated set of colorimeter instructions next to each colorimeter/labquest unit. Students will next measure the absorbance of each solution (their standards and their unknowns) at 635 nm on a colorimeter. The data is recorded on the students Data Table. Interpretation of Results Students should graph their data to develop a standard curve and then use the standard curve to measure the concentration of protein in their unknowns (based on the relationship between absorbance and concentration as detailed in Beer s Law). Students should be able to conclude which of the three companies is accurate in its claim that its sports drink contains the most protein based on their group s results. Ask each team of students to post their results on the board. Discuss the reasons for discrepancies in the results (such as pipetting error, timing errors, limitations of graphing by hand, etc.). Calculate a class average for the concentration of each protein solution (A, B, and C) and determine the standard deviations. After the class shares and analyzes the class data, have students complete the Summary Questions (S-13), the C-E-R (S-14), then the Report (S-15). It is important for students to support their claims of which sports drink has the most protein and to explain the reasoning that the evidence supports the claim. This is the basis for a scientific explanation and, depending on the reasoning used, a scientific argument. Beer s Law should be a part of the students reasoning, and the Summary Questions and C-E-R should help prepare the students to complete their reports. Resist the temptation to give the students the correct answer. In a research laboratory, a scientist would not be able to confirm they had the correct answer, yet scientists are confident in their results. If your students ask you what the right answer is, use that question as an opportunity to discuss the nature of authentic science. You can ask the students how they think scientists handle this element of uncertainty when they conduct their own research. Ideas that may come up in the discussion include: Scientists may repeat an experiment a number of times to confirm their conclusions are reliable. Scientists must carefully record their protocols, procedures and results so they can carefully scrutinize them for consistency. Scientists often share their work and collaborate with others, allowing their methods, results and conclusions to be critiqued and validated. 31 P a g e

32 Teaching the Laboratory Activity The following graph is what the final graph should resemble on the student worksheets. (This graph is provided to assist the educator in evaluating student progress; it is not meant to be shared with the students.) A b s o r b a n c e Line of Best Fit Known Protein Samples Unknown A Concentration (0.015 µg/µl) Unknown C Concentration (0.025 µg/µl) Unknown B Concentration (0.04 µg/µl) Protein Concentration (µg/µl) The green line is the standard curve based on the points obtained from samples #1, 2, 3, 4, and 5, which are the set of protein standards. The blue line is unknown A (with a concentration µg/µl of protein), the red line is unknown C (with µg/µl of protein), and the purple line is unknown B (with µg/µl of protein). Therefore, unknown sample B has the most protein. 32 P a g e

33 Answer Keys to Student Worksheets Macromolecule Scavenger Hunt Key Nucleic Acids 1. List the five monomers of nucleic acids. Adenine (A), Guanine (G), Cytosine (C),Thymine (T), Uracil (U) 2. What role does nucleic acid play in the cell? In the body? Nucleic acids serve as DNA and RNA in the body. DNA codes for all instructions for the cell and by extension, for the body. DNA consists of genes and the genes give instructions for making the proteins. RNA has various roles, including serving as templates for making proteins and carrying the amino acids to make the protein sequence. 3. What happens if the nucleic acid sequence changes in a cell? If the nucleic acid sequence changes, then the protein which is encoded in that sequence can also change. Changes are called mutations. 4. What is the chemical reaction called when two nucleic acid monomers bond together? This is a condensation reaction or a polymerization reaction. Carbohydrates 1. Carbohydrates are divided into two major subgroups. What are those groups and how are they divided? Simple sugar (basically one or a few monomers strung together) Polysaccharides OR complex carbohydrates (many monomers strong together) 2. What roles do carbohydrates play in the cell? In the body? Carbohydrates are structural molecules and they provide energy. They serve as the primary energy source in both the cell and the body. 3. How do plants store carbohydrates? As starch and fiber 4. What foods provide carbohydrates? Grains, cereals, sweets, fruits 5. Many people try to limit carbohydrates. What benefit does this serve? Describe any risks to removing all carbohydrates from the diet. Reducing carbohydrates can reduce calories, so people trying to lose weight often target consuming fewer carbohydrates as part of a weight loss strategy. Removing all carbohydrates from the diet limits energy inputs and removes they body s access to 33 P a g e

34 Answer Keys to Student Worksheets these building blocks (carbs serve as structural components, too), which can hurt the body. Lipids 1. What roles to lipids play in the cell? In the body? Lipids insulate and protect organs, help absorption of some vitamins, and serve as structural molecules. 2. What role do lipids or fat play in food taste and texture? Lipids often carry the molecules that provide flavor to food, so reducing the fat can also reduce food flavor. Lipids also serve important roles in food textures, so textures frequently change when lipids are removed or reduced. 3. What foods provide lipids? Dairy products, meats, oil. 4. Many people try to eliminate or significantly limit lipids in the body. What benefit does this serve? Describe any risks to removing all lipids from the diet. Lipids store energy, so reducing lipids reduces calories. If all lipids were removed from the diet, a person might lose weight by lack the resources to protect organs or absorb some vitamins. Proteins 1. What is the monomer of proteins? How many are there? Amino acids. There are 20 of them. 2. What foods provide proteins? Beans, meats, dark green vegetables 3. What roles do proteins play in the cell? In the body? They serve structural roles and can provide long-lasting energy for cells. They serve as enzymes and muscles in the body. 4. Many people, especially athletes, pay close attention to their protein intake. Would athletes want to increase or decrease their protein intake? Why? Since muscle consists of protein, increasing protein in the diet allows protein to be accessible to build more muscle and to repair muscle. More muscle can help athletes perform better. 34 P a g e

35 Answer Keys to Student Worksheets Roy, Gee and Biv s Worksheet Answers Use the following table to record your additions and subtractions to your test tubes. Test Tube Number Starting Volume (color to be added or subtracted) Amounts Added or Subtracted to the Starting Volume Total Volume at End in microliters (µl) (color of tube) Total Volume at End in milliliters (ml) µl (Red) µl µl (Yellow) µl µl (Blue) µl What is the spectrum that you created? A rainbow 35 P a g e

36 Answer Keys to Student Worksheets for Teachers Summary Questions Answers 1. What is a standard? A standard is a basis for comparison. It is a reference against which something can be evaluated. For example, a ruler is a standard used to measure length (inches or centimeters are the units). In this experiment, our set of protein solutions of known concentrations is the standard (or set of standards) we can use to extrapolate the results of our unknown samples (the unit of measurement is µg/µl). 2. How were standards used for this experiment? In this experiment, our set of protein solutions of known concentrations is the standard (or set of standards). By plotting the absorbance results for this set of standards after performing the Lowry Assay on them, as with the unknown samples, we can use a graph to extrapolate the amount of protein in our unknown samples. 3. Why is the Copper Reagent (Cu Reagent) added to the test tubes? The copper reagent (Cu +2 ), when combined with protein, forms a copper/protein complex. The formation of this is the first step in a two-step reaction, called the Lowry Assay. The copper/protein complex is reduced when Folin-Phenol is added, which ultimately produces a blue-gray colored solution in which the intensity of color is directly related to the amount of protein present in a sample. If protein is not present in a sample, then the copper reagent will not be reduced to Cu +1 and no color reaction will occur. 4. What happened when the Folin-Phenol was added to the test tubes? Explain why. If protein was present in the sample, the solution turned a shade of blue-gray after adding the Folin-Phenol solution. This happened because the copper/protein complex is reduced by Folin-Phenol and this reduction results in a blue-gray color being produced. Note that Folin-Phenol does not react with Cu +2, so if protein is not present in a sample, then the blue-gray color isn t produced (as seen with test tube #1, which never received protein - only water). 5. How does the amount of color in the tube relate to the amount of protein? The intensity of the blue-gray color produced is directly proportional to the amount of protein that was present in the sample (the darker the blue-gray color, the greater the amount of protein contained in the sample). 6. What does the colorimeter do? The colorimeter is an instrument that allows us to quantify the amount of color in a solution. The colorimeter measures the amount of light that is able to pass through a solution (% transmittance) at a given wavelength. It then is able to calculate the amount of light that the sample was able to absorb based on Beer-Lambert s Law. The % 36 P a g e

37 Answer Keys to Student Worksheets for Teachers transmittance and the calculated absorbance are inversely proportional to one another. Therefore, a dark-colored solution will not allow much light to pass through it and will have a lower % transmittance and a higher absorbance than a lighter-colored solution. In this experiment, the amount of blue-gray color produced by the Lowry Assay for a given sample is directly proportional to the amount or concentration of protein within that sample. The colorimeter, therefore, allows us to precisely quantify the amount of protein present in our unknown samples. 37 P a g e

38 Answer Keys to Student Worksheets for Teachers C-E-R suggested answers. Claim (Answer the question, Which sports drink has the most protein in it? ): Answers will vary, but students should find that one protein drink has more protein than the others. In the lab preparation procedure as written, drink B has the most protein. Evidence (Use data from the lab and/or your background reading to support your claim.): Answers will vary, but students should use their graphs here, and mention their standard curves and data collected. They may discuss the color differences they could see when looking at each of the test tubes. Reasoning (Explain how your evidence supports the claim. You may consider using laws or other information to support the connections.) Answers will vary, but students should connect Beer s Law to the protein concentration and absorbance. They may also discuss how a greater concentration of color must mean a greater concentration of protein since the protein is the color. If students had poor results or experimental error, they could also note here that the evidence collected should support the claim, but that they need more evidence because of some concern such as not enough data collected, conflicting class data, errors in the process, etc. 38 P a g e

39 Nutrition Facts Scavenger Hunt S-1 P a g e

40 Macromolecules Scavenger Hunt You will form a team of at least three students. Your team will need to research to answer each of the questions below to learn about the macromolecules which are part of our diets and which serve as the building blocks for all life. There are four macromolecule classes (nucleic acids, carbohydrates, lipids, and proteins). Nucleic Acids 5. List the five monomers of nucleic acids. 6. What role does nucleic acid play in the cell? In the body? 7. What happens if the nucleic acid sequence changes in a cell? 8. What is the chemical reaction called when two nucleic acid monomers bond together? Carbohydrates 6. Carbohydrates are divided into two major subgroups. What are those groups and how are they divided? 7. What roles do carbohydrates play in the cell? In the body? 8. How do plants store carbohydrates? 9. What foods provide carbohydrates? 10. Many people try to limit carbohydrates. What benefit does this serve? Describe any risks to removing all carbohydrates from the diet. S-2 P a g e

41 Macromolecules Scavenger Hunt Lipids 5. What roles to lipids play in the cell? In the body? 6. What role do lipids or fat play in food taste and texture? 7. What foods provide lipids? 8. Many people try to eliminate or significantly limit lipids in the body. What benefit does this serve? Describe any risks to removing all lipids from the diet. Proteins 5. What is the monomer of proteins? How many of them are there? 6. What foods provide proteins? 7. What roles do proteins play in the cell? In the body? 8. Many people, especially athletes, pay close attention to their protein intake. Would athletes want to increase or decrease their protein intake? Why? S-3 P a g e

42 Name Roy, Gee, and Biv s Micropipette Challenge Gee, Roy, and Biv are having problems with their science lab. Their teacher is asking them to construct a model of a spectrum, but none of them have a clue as to what a spectrum is, let alone how to make one. Use the following table and the directions that follow to help them by constructing your own spectrum. Use the following table to record your additions and subtractions to your test tubes. Test Tube Number Starting Volume (color to be added or subtracted) Amounts Added or Subtracted to the Starting Volume Total Volume at End in microliters (µl) (color of tube) Total Volume at End in milliliters (ml) µl (Red) 2 0 µl µl (Yellow) 4 0 µl µl (Blue) 6 0 µl Place a in each box as you complete the steps below. Setting up your tubes: 1. Label the six test tubes at your station, Put 760 µl of red water into test tube number Put 880 µl of yellow water into test tube number Put 1000 µl of blue water into test tube number 5. Constructing ROY s Spectrum: Make sure you record your actions in the table above. 5. Take 160 µl from test tube number 1 and put it into test tube number Take 160 µl from test tube number 1 and put it into test tube number Take 160 µl from test tube number 3 and put it into test tube number Take 280 µl from test tube number 3 and put it into test tube number Take 280 µl from test tube number 5 and put it into test tube number Take 280 µl from test tube number 5 and put it into test tube number 6. Crunching the numbers: 11. Calculate the total volume in each tube and record your answer in the table. 12. Convert your units from µl (microliters) to ml (milliliters). Hint: 1000 µl = 1 ml 13. What is the spectrum that you created? Hint: 1000 µl= 1 ml S-4 P a g e

43 The Power Drink Challenge: Lab Background Background Information: Scientists sometimes measure how much of something there is in liquids. For example, they may measure the amount of lead levels in drinking water or the amount of dissolved oxygen in the Chesapeake Bay. The amount of a substance in a solution is often expressed as weight or volume. Concentration is an expression of how much of one substance is dissolved in another substance. It always has one unit divided by another. For example, if I use 3 cups of sugar in 1 gallon of lemonade, the amount of sugar is 3 cups but the concentration of sugar is 3 cups per gallon. The Nutrition Facts Label on food and drinks contains information about the nutrients in the product. Refer back to your completed Nutrition Facts Scavenger Hunt. 1. How many grams of total carbohydrates are in your food or drink? (Your answer should include units.) What two components make up the total carbohydrates? 2. How many grams of protein per serving are in this food or drink? (Your answer should include units). 3. What is the CONCENTRATION of protein in this food or drink? (Your answer should include units). Laboratory Challenge: Several companies are competing to produce a new product, The Power Drink. It is a high-protein drink for athletes to improve their physical performance. Three companies advertise that they produce a drink with the highest concentration of protein. Sharon works for an independent testing agency and has been hired to settle the dispute among the companies. She will test the concentration of protein in each drink. You and your partner can help Sharon find out which of the three companies can actually claim they make the drink with the highest amount of protein. Your job is to determine the concentration of protein in the three drinks. The results from each team will be compared and you will present your conclusions to the companies. You will have to defend your conclusions, especially to the companies that lost. How will you see the protein? Protein is colorless you cannot look at each sample to see the amount of protein in it. But there is a chemical reaction that can make protein turn a blue color - the darker the color, the greater the amount of protein in the sample. It is called S-5 P a g e

44 The Power Drink Challenge: Lab Background the Lowry Assay. Sharon has decided to use the Lowry Assay to help her determine the amount of protein in the different sports drinks. How does the Lowry Assay work? The Lowry Assay uses chemical reactions to add color to protein in solutions. The Lowry Assay must be done in two steps since it involves two chemical reactions. (A chemical reaction is a chemical change that forms new substances). Reaction 1: Protein (colorless) + = Copper Reagent Wait 5 minutes Cu +2 /protein Complex (mostly colorless) The Copper (Cu) Reagent reacts with the colorless protein in the solution to form a copper/protein solution, which is still mostly colorless. Reaction 2: Cu +2 /protein Complex ( mostly colorless) + Folin-Phenol Wait 5 = minutes The Folin-Phenol reacts with the Copper/Protein Complex and a dark blue color is produced in direct proportion to amount of protein present. The more protein, the darker the solution. 3. What is the purpose of the Lowry Assay? 4. Why do you need to do the assay in two steps? S-6 P a g e

45 Materials The Power Drink Challenge: Lab Procedure You will find the following tubes and samples at your workstation: Three unknown sports drink samples, labeled A, B, and C, in conical tubes. One conical tube labeled Stock Protein (0.5 µg/µl) that contains protein of a known concentration. You will use this to make your set of protein standards. Eight empty test tubes: The ones that you will label #1-5 will be used to mix known concentrations of protein and distilled water to create a set of standards. The ones that you will label A, B, and C will be used for the unknown samples A, B, and C. One conical tube labeled Cu Reagent that contains the copper reagent. One conical tube labeled Folin-Phenol that contains the Folin-Phenol reagent. One conical tube labeled dh2o that contains distilled water. 8 square plastic cuvettes, labeled #1-5, and A, B, and C, to be used with the colorimeter (in a cuvette rack) 1. What is the goal of this lab? 2. What assay are you going to use? 3. Describe the set of standards you will be making. Protocol Prepare Protein Standards: 1. Use a Sharpie to label your test tubes A, B and C, and In the next two steps, you will be preparing five different concentrations of protein to use as standards. How will a set of standards help you figure out how much protein is in the sports drinks? Write your answer below. 3. First, add different amounts of dh2o to test tubes #1-5 (see Column 2 in the data table for specific amounts). S-7 P a g e

46 The Power Drink Challenge: Lab Procedure 4. Next, add different amounts of Stock Protein (0.5 µg/µl) to test tubes #1-5 (see Column 3 of the data table on page S-11 for specific amounts). Calculate Concentration of Standards 5. Calculate the amount of protein (in µg) in each test tube. Write your answers in Column 4 in your data table on page S-11. Your answer should be in µg. 6. Calculate the final volume of each test tube in µl and fill in Column 5 in your data table. Your answer should be in µl. 7. Calculate the final concentration of standards and fill in Column 6 in your data table. Your answer should be in µg/µl. Prepare Unknown Sports Drink Samples 8. Add 1000 µl of each unknown sample (A, B, and C) from the conical tubes to their corresponding test tubes labeled A, B, and C. These are the sports drink samples for which you want to determine the protein concentrations. Perform the Lowry Assay 9. Add 1000 µl of Cu Reagent from the conical tube to each of your test tubes (# 1-5, and A, B, and C). Gently pipette up and down at least five times to mix each solution. 10. Incubate the test tubes at room temperature for 5 minutes. 11. Add 200 µl of Folin-Phenol reagent to each of your test tubes (# 1 5, and A, B, and C). Gently pipette up and down at least five times to mix each solution. 12. Incubate the test tubes at room temperature for 5 minutes. How do I calculate concentration? Concentration is a measure of amount of solute (in this case protein) in a solvent (in this case water). Analysis of the Protein Standards and Unknown Samples Analysis using a colorimeter: The colorimeter measures absorbance, which is the amount of light absorbed by the color in the test tubes - the darker the color, the higher the absorbance value. 1. You need to know concentration of the stock protein solution you added to each tube above (hint- look at column 3 heading in your data table and make sure to write your units) 2. Next, you need to figure out the amount (in µg) of protein you added to each test tube. For example in Test Tube 2, you added 50 µl of 0.5 µg/µl. 50 ul X 0.5 ug/ul = 25ug of protein. 3. Next you need to figure out the final concentration in the test tube. This will be the amount of protein (in µg) over the final volume in the test tube (in µl). For example, in Test Tube 2 you had 25 ug protein/1000 ul or ug/ul. S-8 P a g e