The
Crucial Concentration
Investigating Unknown Quantities of Protein Using the Lowry Assay
Maryland Loaner Lab Teacher Packet
www.towson.edu/cse
Version Aug16_kny
Crucial Concentration
Table of Contents
TEACHER MATERIALS
Materials and Supplies
Correlation to Standards
Introduction and Overview for Teachers
Background Information for Teachers
Facilitation Guide
Pre-Laboratory Information for Teachers
Laboratory Preparation for Teachers
Teaching the Lab Activity
Answer Keys to Student Worksheets
3
6
7
9
11
16
22
27
30
STUDENT MATERIALS
Nutrition Facts Scavenger Hunt
Macromolecules Scavenger Hunt
Roy, Gee, and Biv’s Micropipette Challenge
Lab Background for Students
Lab Procedure for Students
Data Table
Graph
Summary Questions
C-E-R Chart
Report It!
Colorimeter Demonstration Worksheet
S-1
S-2
S-4
S-5
S-6
S-11
S-12
S-13
S-14
S-15
S-16
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Crucial Concentration
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 PreLab 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 ActivityMicropipette Challenge.
Each group gets a red,
yellow and blue tube.
Yes. Return unused
portions.
Bag labeled “Solution
1and Solution 2 for
Lab Activity”
- 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 CO3 (sodium
carbonate)
Sol 2 to be added to Sol 1
by teacher, see prep
directions on page 15.
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 2CO3
(sodium carbonate)
Bag of empty tubes
labeled “Cu Reagent”
10 empty (15ml) conical tubes
labeled “Cu Reagent”
Bag containing 10
empty 15ml conicals
labeled “FolinPhenol” for FolinPhenol Reagent”
Bag of tubes of
empty distilled water
labeled “dH2O tubes”
10 (15ml) empty conical tubes
labeled “Folin-Phenol”
10 (15ml) conical tubes labeled
“dH2O”
See “Laboratory
Preparation for Teachers”
for details on using Na 2 CO3
(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.
1 conical tube per group
1 tube of distilled water for
each of 10 groups
Clean and return
If used, do not return
Yes, empty tubes rinsed
and dried
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Crucial Concentration
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)”
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
Yes, empty tubes rinsed
and dried
Return inside insulated
bag
- Teacher Demo Set (8 (50 ml)
conicals)
Return Demo Set filled
(do NOT empty).
1000 µl
micropipettes
1000 µl tips
10
- Set of 8 (50 ml) conicals of
blue water of known
concentration (6) and
conicals of blue water of
unknown concentration (2)
1/group
Yes, unused tips
200 µl micropipettes
200 µl tips
10
5 boxes
Teaspoons
Insulated Bag &
freezer pack
Tupperware
container labeled
“Glass test tubes”
10
1
1 box per 2 groups (tips are
blue)
1/group
1 box per 2 groups (tips are
yellow)
1/group
~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
tubes
Yes
Cuvette sets
Cuvette racks
10 sets of 8 cuvettes each,
labeled “1-5, A, B, C”
10
Yes, empty tubes
rinsed and dried
Yes
Test Tube Rack
Disinfectant Wipes
10
1 bottle
Colorimeter and
LabQuest Data
Unit
3
8 per group to use in
colorimeter
1 per group to hold
cuvettes
1 per group
Please wipe down
pipettes
Each kit will have 3 kits
to be shared among
student groups
5 boxes
Yes
Yes
Yes, unused tips
Rinse, dry, and return
Return
Yes
Yes
Yes
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Crucial Concentration
Materials and Supplies
Lab flashlight
1
Kimwipes
3-6
For teacher colorimeter
demo
Place one by each
colorimeter to clean
smudges from cuvettes
Materials provided by Teacher:
Description
Quantity
1-2 bag lentils (red preferred,
but any color lentil or a
substitute dry bean will work)
1 4 lb bag of white sugar
Beakers or cups (may be
disposable)
Provide roughly ½ c sugar
per lab group
4/lab group
~2/group
Graduated Cylinder (10ml)
Graduated Cylinder (1000 ml)
Parafilm
1
1
Enough to cover beaker
to mix solution (optional)
10
10ml of 2N Folin-Phenol
for each class set
60 ml Distilled Water per class
set
NaOH (sodium hydroxide)
Reusable 50ml conical tube
or beaker
Yes
Comments
Provide roughly 1/3 c
Used with “What’s in Your Food?”
dried lentils per lab group
Food containers and labels
Disposable Cups
2N Folin-Phenol Reagent
Yes
For each class set10 ml for Folin-Phenol
Solution and 50ml for
Solution 1
0.8 g per class set
1 conical tube or beaker
Used with “What’s in Your Food?”
Use 1 for sugar, 1 for lentils, then 2
empty/lab group for “What’s in Your
Food?”
Can have students bring in their own food
packages and labels.
Used to measure Cu Reagent
Used to prepare Solution 1
Used to prepare Solution 1
1 per group – waste container for lab
2N Folin-Phenol Reagent can be ordered
online at
http://www.sigmaaldrich.com/catalog/pro
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
Used to make Solution 1. 1N NaOH can be
ordered at
http://www.sciencecompany.com/P15983C670.aspx?utm_source=google&ut
m_medium=shop&utm_campaign=prod
Cost: $23.50 for 500 grams
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.
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Crucial Concentration
Standards Chart
Maryland Science Core Learning Goals
Goal 1.0 Skills and Processes
1.3
The student will carry out scientific investigations effectively and employ the
instruments, systems of measurement, and materials of science appropriately.
1.4.4 The student will determine the relationships between quantities and develop the
mathematical model that describes these relationships. *
1.1.5 The student will explain factors that produce biased data (incomplete data, using
data inappropriately, conflicts of interest, etc.).
1.2.1 The student will identify meaningful, answerable scientific questions.
1.2.6 The student will identify appropriate methods for conducting an investigation
(independent and dependent variables, proper controls, repeat trials, appropriate sample
size, etc.).
1.2.8 The student will defend the need for verifiable data.
1.3.1NTB The student will develop and demonstrate skill in using lab and field equipment to
perform investigative techniques. *
1.3.2 The student will recognize safe laboratory procedures. *
1.3.3NTB The student will demonstrate safe handling of the chemicals and materials of science.
*
1.3.4NTB The student will learn the use of new instruments and equipment by following
instructions in a manual or from oral direction. *
1.4.1 The student will organize data appropriately using techniques such as tables, graphs,
and webs. (for graphs: axes labeled with appropriate quantities, appropriate units on axes,
axes labeled with appropriate intervals, independent and dependent variables on correct
axes, appropriate title)
1.4.2 The student will analyze data to make predictions, decisions, or draw conclusions.
1.5.4 The student will use tables, graphs, and displays to support arguments and claims in
both written and oral communication.
1.6.5 The student will judge the reasonableness of an answer.
1.7.5 Students will investigate career possibilities in the various areas of science.
Goal 4.0 Concepts of Chemistry
4.1.2 The student will gather and interpret data related to physical and chemical properties
of matter, such as density and percent composition. (constructing data tables, graphing linear
relationship, appropriate technology to analyze data) *
* Italicized CLG’s are the primary focus of the laboratory activity. The other indicators are
addressed, but can also be used as a guide to determine the focus of the lesson plans for the
pre-lab and post-lab extension activities.
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Crucial Concentration
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
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Crucial Concentration
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.
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Crucial Concentration
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.
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Crucial Concentration
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.
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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-
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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
(http://school.discoveryeducation.com/foodscience/college_resources.html) 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 10-30 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.
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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 (S13), 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.
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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 BeerLambert 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.
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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.
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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
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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
1 1000 µl micropipette
1 200 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
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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.
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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
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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
Unknown 2
0
1
5
10
15
20
50
50
50
50
50
50
50
50
0
0.02
0.1
0.2
0.3
0.4
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.
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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.
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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 Na2 CO3 (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 1.
2. 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 2.
3. 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.
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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 0.015 µg/µl BSA, “Unknown B” has 0.040 µg/µl
BSA, and “Unknown C” has 0.025 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).
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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,
Fig. 2. Colorimeter
and C) into their cuvettes (labeled #1-5, and A, B,
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 0.000 or 0.001 (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
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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
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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.
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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
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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.
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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.)
0.45
0.4
Line of
Best Fit
A 0.35
b
s 0.3
o
0.25
r
b 0.2
a
n 0.15
c
e 0.1
Known Protein
Samples
0.05
0
0
Unknown A
Concentration
(0.015 µg/µl)
0.02
Unknown C
Concentration
(0.025 µg/µl)
0.04
0.06
0.08
0.1
0.12
Protein Concentration (µg/µl)
Unknown B
Concentration
(0.04 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 0.015 µg/µl of protein), the red line is
unknown C (with 0.025 µg/µl of protein), and the purple line is unknown B (with 0.040 µg/µl of protein).
Therefore, unknown sample B has the most protein.
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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
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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.
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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)
Total Volume at End
in microliters (µl)
(color of tube)
Total Volume at
End in milliliters
(ml)
1
760 µl (Red)
-160
-160
440
.44
2
0 µl
+160
+280
440
.44
3
880 µl (Yellow)
-160
-280
440
.44
4
0 µl
+160
+280
440
.44
5
1000 µl (Blue)
-280
-280
440
.44
6
0 µl
+160
+280
440
.44
13.
Amounts Added or
Subtracted to the Starting
Volume
What is the spectrum that you created? A rainbow
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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 %
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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.
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Crucial Concentration
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.
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Crucial Concentration
Nutrition Facts Scavenger Hunt
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Crucial Concentration
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.
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Crucial Concentration
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?
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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)
1
760 µl (Red)
2
0 µl
3
880 µl (Yellow)
4
0 µl
5
1000 µl (Blue)
6
0 µl
Amounts Added or
Subtracted to the Starting
Volume
Total Volume at End
in microliters (µl)
(color of tube)
Place a  in each box as you complete the steps below.
Setting up your tubes:
1.
Label the six test tubes at your station, 1-6.
2.
Put 760 µl of red water into test tube number 1.
3.
Put 880 µl of yellow water into test tube number 3.
4.
Put 1000 µl of blue water into test tube number 5.
Total Volume at
End in milliliters
(ml)
Hint: 1000 µl= 1 ml
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 2.
6.
Take 160 µl from test tube number 1 and put it into test tube number 6.
7.
Take 160 µl from test tube number 3 and put it into test tube number 4.
8.
Take 280 µl from test tube number 3 and put it into test tube number 2.
9.
Take 280 µl from test tube number 5 and put it into test tube number 4.
10.
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?
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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
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)
+
=
Wait 5
minutes
Copper Reagent
Cu+2/protein Complex
(mostly colorless)
(colorless)
Reaction 2:
Folin-Phenol
=
+
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.
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?
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The Power Drink Challenge: Lab Procedure
Materials
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 1-5.
2. 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
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.
How do I calculate concentration?
Concentration is a measure of amount of solute (in this case protein) in a solvent (in
this case water).
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
0.025 ug/ul.
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.
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.
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The Power Drink Challenge: Lab Procedure
13. Transfer 2 ml of each of your samples from the glass test tubes to the small plastic
cuvettes. Make sure to put your samples in the appropriately labeled cuvette. (Sample
#1 should go into the cuvette labeled “1”). Use a NEW micropipette tip for each sample.
14. Make sure your 8 cuvettes (samples 1-5, and A, B, and C) are in the cuvette rack and
take them to the colorimeter. Your teacher will demonstrate how to use the
colorimeter. Groups must take turns on the colorimeter and should work as quickly as
possible to allow the next group to use it.
15. Colorimeter instructions:
a. Clean cuvette “1” with a kimwipe and gently place it into the colorimeter with the arrow
on the top of the cuvette slot on the colorimeter pointing to the clear side of the
cuvette. DO NOT force it down; gently insert only as far as it easily goes.
b. Press the “CAL” (calibrate) button on the colorimeter and hold it until the red LED
begins to flash (this usually takes less than one second). When the reading on the
LabQuest unit is either 0.000 or 0.001, you have calibrated the colorimeter for the rest
of your cuvettes.
c. Remove cuvette “1”.
d. Clean cuvette “2” and insert it into the colorimeter. Do NOT hit “CAL” anymore; that is
only done for the first sample to calibrate the colorimeter for your samples. Record the
absorbance on your data table. (It may take a second or two for the reading to
stabilize). Gently remove the cuvette.
e. Repeat above step (step d) for samples #3-5 AND for each of your unknown samples (A,
B, and C). Don’t forget to record each absorbance reading on your data table.
Graphing Your Standards as Absorbance versus Protein Concentration
16. Plot absorbance versus protein concentration data for the five protein standards
(samples #1-5) on the graph paper at the end of your packet. (Do NOT plot your
unknowns yet).
17. Draw a “best-fit” line through the data points. (A best fit line is a STRAIGHT line that
best describes the trend of the data. It will not ‘connect the dots’.)
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The Power Drink Challenge: Lab Procedure
Determine Concentration
of Unknown Samples
18. To determine the
Unknown
concentration of
Sample
protein in each of
your unknown
samples, find its
absorbance on
the y-axis and draw a horizontal line across
the graph until you reach your best-fit line.
Then, draw a vertical line from your line of
best-fit down to the x-axis. The point where
this vertical line intersects the x-axis will tell
you the concentration of the unknown sample.
Absorbance
Protein Concentration (ug/ul)
a. Find the concentration of Unknown A; write the result in Column 6 of your data table.
b. Find the concentration of Unknown B; write the result in Column 6 of your data table.
c. Find the concentration of Unknown C; write the result in Column 6 of your data table.
S-10 | P a g e
Crucial Concentration Data Table
DATA TABLE
Column 1
Test Tube
Column 2
Amount
of Water
to Add
(µl)
Column 3
Column 4
Column 5
Column 6
Column 7
Amount of Amount of
Stock Protein protein in
to add (µl)
each test
Stock
tube (µg)
protein
concentratio
n is 0.5 µg/µl
0 µl
Volume
in Tube
(µl)
(add
columns
2 and 3)
Concentration
(µg/µl)
Absorban
ce
1
1000 µl
2
950 µl
50 µl
3
900 µl
100 µl
4
850 µl
150 µl
5
800 µl
200 µl
Unknown A
1000 µl
Unknown B
1000 µl
Unknown C
1000 µl
Solute/Solvent
S-11 | P a g e
Crucial Concentration Graph
Absorbance (This measurement does not have units)
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
Protein Concentration
What are your units? Write them here _______________
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Crucial Concentration Lab Summary Questions
Lab Summary Questions
1. What is a standard?
2. How were standards used for this experiment?
3. Why is the Copper Reagent (Cu Reagent) added to the test tubes?
4. What happened when the Folin-Phenol was added to the test tubes? Explain why.
5. How does the amount of color in the tube relate to the amount of protein?
6. What does the colorimeter do?
7. What were the protein concentrations of the unknown sport drink samples? Which one
had the highest protein concentration?
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Crucial Concentration
Claim-Evidence-Reasoning Chart
After analyzing the concentration of protein in each of the sports drinks using Lowry’s Assay,
the food scientist would make a claim and support that claim with evidence and reasoning in a
report of his or her findings. This forms the basis for a scientific explanation or argument,
depending on the actual information provided in the discussion. The skills of writing and
developing scientific explanation and arguments are very important.
Claim (Answer the question, “Which sports drink has the most protein in it?”):
Evidence (Use data from the lab and/or your
background reading to support your claim.):
Reasoning (Explain how your evidence
supports the claim. You may consider using
laws or other information to support the
connections.):
S-14 | P a g e
Crucial Concentration
Report it!
Develop a report to share the findings with consumers. You must include the information
noted below, but you can choose to use any format you choose. For example, you may wish to
write an article for a newspaper or magazine, develop a radio public service announcement or a
video presentation, or prepare a poster or other visual presentation.
Required Information:
1.
2.
3.
4.
5.
6.
7.
8.
What were you testing and why? (i.e., Why did you do this test?)
How did you ensure that the test was unbiased and fair?
What process did you use in this investigation?
What data did you collect?
What claim are you making?
What evidence do you have to support your claim?
Why does that evidence support your claim?
What recommendations do you have, now that this information is available?
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Crucial Concentration
Colorimeter Demonstration Worksheet
The colorimeter works by shining a light through a sample and assigning a numeric value to the
intensity of color. In this demonstration, you will serve as the colorimeter by assigning a value
between 1-10 to each of the samples. Data below allows you to calculate the concentration of
each of the known samples. How will you figure out the concentrations of the two unknown
samples?
Conical
Number of
drops of
blue dye
Volume of
water added
(ml)
A
B
C
D
E
F
Unknown 1
Unknown 2
0
1
5
10
15
20
50
50
50
50
50
50
50
50
Concentration
(drops/ml)
Use the space below to create graph your color intensity (no units) and your concentrations
(drops/ml). What goes along the x axis? Along the y axis? Remember titles and labels!
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