TEACHER GUIDE to Lab Investigation: Protein Fingerprinting
BIG IDEA: Mutations in an organism's DNA can change its characteristics, and these characteristics can help
the organism to survive and reproduce. Sometimes, organisms can change so much over many generations that
their offspring become a new species. For example, the great-great-great-great grandparents (imagine many
more 'great's) of cows and goats were actually brother and sister. As the brother and sister, each with their own
DNA, proteins, and characteristics, went their separate ways and had their own babies, and the babies had their
own babies, and so on - one set of great-great-great grandchildren eventually had cow babies and the other had
goat babies. Even though their ancestors were related, they have evolved into two different species that can no
longer reproduce with each other.
Although some of their characteristics became different, some cow and goat characteristic stayed the same as
the characteristics of their ancestors. Therefore, some of their DNA and proteins will be very different, and
some will be the same.
• Picture a cow and a goat. What characteristics do cows and goats have in common? List as many as you can:
Students will come up with the obvious characteristics, such as both are mammals, four legged, covered
in fur, herbivores, mostly graze on grass, etc. They may also come up with that both are ungulates (eventoes, rather than odd like horses), and they are ruminates with four chamber stomachs.
• What characteristics are different between cows and goats? List as many as you can:
Size if course, a cow can be almost 10X the size of a goat, cows are pickier eaters, goats are climbers.
In this activity, you will create protein fingerprints of cows, goats, and several other animals to determine how
their ancestors were related. First, think about the animals' characteristics and how they might be related.
Display your thinking as a phylogenetic tree.
How to design a phylogenetic tree: Here is an example phylogenetic tree of five organisms: fruit flies, worms,
horses, turkeys, and frogs. These organisms have some characteristics in common (they all are made of many
cells, they all have mouths, etc.), and some characteristics that are different (some have hair, some have
feathers, some have eyes and some don't, etc.). On the phylogenetic tree, the simpler the animal, the closer you
diagram the animal to the common ancestor (like the worm). The more complex the animal, the farther it is
away from the common ancestor (like the horse). Finally, the more closely related two animals are, the closer
they are on the phylogenetic tree (fruit flies are more closely related to worms than to frogs, turkeys, or horses).
Sample phylogenetic tree:
Horse
Turkey
Frog
Fruit fly
Worm
Common
ances tor
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Your challenge is to design a phylogenetic tree of the organisms whose protein fingerprints you will examine.
Phylogenetic tree: Most students will draw the tree like this, placing fish and squid on a separate branch
because they are both water animals.
The tree should look more like this:
Cow
Turkey
Squid
Fish
Squid
Fish Turkey Cow
In this activity we will look at the protein profiles of these animals to determine if we can observe similarities
and correlate these similarities to their evolutionary relationship to each other. In the students’ version of the
above tree you would expect the squid and fish to share protein similarities compared to cow and turkey
who should share proteins relative to each other.
What is protein gel electrophoresis? First, proteins are what the students are going to examine with the gel.
Second, the gel you will use is called agarose, a carbohydrate isolated from seaweed. Third, 'electro' means
electricity. Finally, 'phorese' is a Greek word meaning 'to carry'. Students will use electricity to carry the
proteins through the agarose gel. Electricity will carry the proteins because, in this experiment, the proteins are
negatively charged. If the proteins were neutral (no charge), electricity would have no effect on them.
How is protein gel electrophoresis useful? It's like making spaghetti- when you finish boiling the noodles, you
strain the noodles away from the water. The noodles don't do through the holes in the strainer because they're
too big. If they were smaller, the noodles could fit through the holes. Proteins are like different sized noodles
and the agarose gel is like a strainer, filled with little holes. Proteins that are small moves easily through the
holes in the agarose gel, while larger proteins move more slowly, getting stuck in the holes in the gel. Thus,
protein gel electrophoresis allows students to spread out the proteins from different bovine tissues, creating a
protein fingerprint that serves as a barcode to identify that type of cell or tissue.
Almost every cell contains the DNA to make every protein the cell needs. Once they've run their gels, students
will be able to observe different protein fingerprints for each animal and correlate the protein fingerprints to the
evolutionary relationships.
Materials/Equipment Needed
For the class:
• hot water bath (70°C)
• hot water bath or heat block (95°C)
• 2 thermometers
• microwave or hot plate
• 3% agarose in Tris-Glycine buffer (about 35 ml per group, depending on size of gel tray)
- agarose powder
- Tris base
- glycine
- distilled water
• Tris-Glycine-SDS buffer (about 250 ml per group, depending on size of electrophoresis apparatus)
- Tris base
- glycine
- sodium dodecyl sulfate (SDS)
- distilled water
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• Coomassie blue stain (about 50 ml per group, depending on size of staining trays)
- Coomassie blue
- methanol
- glacial acetic acid
- distilled water
• Destain (about 100 ml per group, depending on size of staining trays)
- methanol
- acetic acid
- distilled water
• Plastic wrap
• 4 different animal tissues (we usually send out cow, turkey, fish and squid)
For each student group (preferably groups of 4):
• horizontal gel electrophoresis apparatus, electrodes, power supply
• micropipet with 4 micropipet tips [or 4 transfer pipets]
• 4 microfuge tubes with 500 µl sample buffer
- Tris base
- sodium dodecyl sulfate
- bromophenol blue
- glycerol
• 4 empty microfuge tubes
• staining tray
• paper towel or kleenex
Science Background
Sample buffer: The sample buffer contains several ingredients useful for extracting proteins and conducting gel
electrophoresis. The Tris is a buffer, which helps keep pH constant during the experiment. SDS (sodium
dodecyl sulfate, also known as lauryl sulfate) is a detergent that dissolves cell and nuclear membranes by
breaking down lipids (fat), as well as unfolding the proteins in the sample. The bromophenol blue adds color to
the solution so students can see the samples as they load them into the gel and determine when the gel has run far
enough (the dye is 6-8 cm from the wells). Glycerol is a very dense liquid that makes the samples dense so they
sink to the bottom of the wells in the gel.
Tris-Glycine-SDS buffer: This solution helps: keep the pH constant during gel electrophoresis (Tris); conduct
electricity (glycine does this because it's charged); and keep the proteins unfolded and negatively charged so they
can move through the gel toward the positive electrode (SDS does this because it is negatively charged and sticks
to the proteins). Heating the proteins prior to loading them in the gel also helps unfold the proteins so they can
move through the gel (Step 6 in preparing muscle samples).
Electrophoresis: Electrophoresis is an oxidation/reduction reaction. H20 splits into H+ and OH-; H+ travels to
the negative electrode (black) and OH- to the positive electrode (red). At the negative electrode, H+ gains an
electron (is reduced) and becomes hydrogen gas [2H+ and 2 electrons become H2 (gas)]; at the positive electrode,
O2- loses two electrons (is oxidized) and becomes oxygen gas [2O2- becomes O2 (gas) and 4 electrons]. As H+ is
reduced at the negative electrode, it leaves behind the base, OH-, turning negative end basic. As O2- is oxidized
at the positive electrode, it leaves behind the acid, H+, turning the positive end acidic. You'll use a buffer (Tris)
to neutralize the acid and base.
Staining: While the gel is running, the students will only be able to observe the bromophenol blue. They will
not be able to see any proteins (bromophenol blue doesn't stain proteins) until they stain the gels with Coomassie
blue, a protein dye.
Teacher Note
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This activity takes 2-3 days, depending on your class schedule. Luckily, there are several points at which the
experiment can be stopped. For example, the gels can be stored at several points throughout the experiment,
even over a weekend: 1. when they are poured but not yet loaded (store wrapped in refrigerator), 2. when they
are run but not yet stained (store wrapped in refrigerator), and 3. when they are stained and in process of
destaining (store in destain covered with plastic wrap at room temperature for up to 2 days, any longer and the
proteins won't be stained enough).
Here are example timelines for conducting the protein electrophoresis experiment. You can determine what's
appropriate given your schedule (whether you have a block period), your timing (do you have 2 or 3 days to do
the activity?), and your supplies (1 or 2 class sets of electrophoresis equipment).
Timelines for protein gel electrophoresis
Loading gel/
Pouring gel
Preparing
samples
No block
in advance by Day 1
periodteacher
(15 minutes)
done in 2
days
[1 class set]
No block
Day 1
Day 2
period(10 minutes), (15 minutes)
done in 3
then store
days
overnight in
[1 class set]
plastic wrap
or ziplock
bag
No block
Day 1
Day 1
period(10 minutes) (15 minutes)
done in 3
days
[2 class sets]
Block perioddone in 2
days
(be sure to
start the
experiment
on block day)
[1 class set]
Day 1
(10 minutes)
Day 1
(15 minutes)
Running gel
Staining gel
Day 1
(30 minutes)
Day 1
(30-90
minutes),
students start
staining
Day 2
(30-90
minutes),
students start
staining
Day 2
(30 minutes)
Day 1start running,
finish
running in
the next class
period while
the next class
starts the
activity (store
in plastic
wrap
overnight)
Day 1
(30 minutes)
Advance Preparation for Protein Electrophoresis Lab
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Destaining
gel
Day 1
(overnight)
by teacher
Analyzing
data
Day 2
(30-50
minutes)
Day 2
(overnight)
by teacher
Day 3
(30-50
minutes)
Day 2
(30-50
minutes),
students stain
gels for entire
period then
start destain
Day 2
(overnight),
students start
destain
Day 3
(30-50
minutes)
Day 1
(30-90
minutes),
students start
staining
Day 1
(overnight)
by teacher
Day 2
(30-50
minutes)
1. Make all of the solutions listed in the recipes above.
2. Melt the agarose, and store melted in a 70°C water bath or on a hot plate.
3. Set up electrophoresis equipment.
4. Set heat block or second water bath to 95°C.
5. Assemble set of materials for students: 4 microfuge tubes with sample buffer, 4 empty microfuge tubes, 4
pipet tips, and one micropipet.
Analysis
1. Compare the protein fingerprints from the different tissues. What can you conclude about what proteins each
type of cell makes?
The protein fingerprints for each type of tissue should look different. Each type of cell makes different
types of proteins, although some proteins each cell makes might be the same because some cells have to do
the same jobs (e.g. cellular respiration).
2. Which tissues had the most similar proteins? Which tissues had the most different proteins? Why do you
think so?
The more similar the types of cells (e.g. skeletal muscle and cardiac muscle), the more similar their
proteins will be. The more different the types of cells (e.g. skeletal muscle and liver), the more different
the proteins in those cells will be.
3. Why would proteins from different types of cells look different? Why would they look the same?
Since each cell has specific functions to perform, the more similar the cells' functions are with other cells,
the more similar their proteins will be.
Questions
1. What is a protein fingerprint?
A protein fingerprint is the pattern of proteins made by a specific type of cell or tissue, it's like a barcode
for identifying that cell or tissue.
2. Does each type of cell make the same type of proteins? Why or why not?
Each type of cell does not make the same type of proteins because each cell has a different job to do. Each
cell only makes the proteins it needs.
3. Why are the proteins loaded in the gel near the negative electrode?
The proteins are negatively charged, so they will move toward the positive electrode. If they were loaded
near the positive electrode, they would move off the end of the gel. [Note: The proteins in this experiment
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are negatively charged because SDS, which is negatively charged, is in the gel buffer and sticks to proteins,
making them negative as well.]
Recipes and Background Information
5X stock of Tris-Glycine buffer
Combine 15.1 g Tris base and 94 g glycine with water to make a total volume of 1 liter. Dilute this 1:4 with
water (i.e. 100 ml stock with 400 ml water to make a total 500 ml of 1X Tris-Glycine buffer). Store in a sealed
bottle at room temperature indefinitely.
3% agarose in Tris-Glycine buffer
In a 250 ml Pyrex bottle, combine 3.75 g agarose with 125 ml Tris-Glycine buffer - DO NOT USE TRISGLYCINE-SDS BUFFER FOR THIS! You'll end up with a giant bubbly mess. Microwave uncovered for 1
minute at a time until agarose is dissolved, being careful not to let the agarose boil over on the microwave or on
you! Store loosely covered at room temperature until solidified, then tightly cover to store indefinitely. Be sure
to remove cover before you microwave to dissolve the agarose again.
5X stock of Tris-Glycine-SDS buffer
Combine 15.1 g Tris base, 94 g glycine, and 50 ml 10% SDS (5 g SDS with 45 ml water) with water to make a
total volume of 1 liter. Dilute this 1:4 with water (i.e. 100 ml stock with 400 ml water to make a total 500 ml of
Tris-Glycine-SDS buffer). Store in a sealed bottle at room temperature indefinitely.
Coomassie blue stain
For each liter of stain, combine 450 ml water, 2.5 g Coomassie blue, 450 ml methanol, and 100 ml glacial acetic.
Store in a sealed bottle at room temperature indefinitely.
Destain
For each liter of destain, combine 600 ml water, 300 ml methanol, and 100 ml glacial acetic acid. Store in a
sealed bottle at room temperature indefinitely.
1.0 M Tris-Cl (pH 6.8)
Combine 60.5 g Tris base with about 350 ml water. Add enough HCl to give the solution a pH of 6.8. Add
enough water to make the total volume of the solution 500 ml (0.5 liter). Store in a sealed bottle at room
temperature indefinitely.
Sample buffer
To make 100 ml sample buffer, combine 10 ml 1.0 M Tris-Cl (pH 6.8), 20 ml 20% SDS, 0.1 g bromophenol
blue, 20 ml glycerol, and water to make a total volume of 100 ml. This is actually a 2X recipe, but the sample
buffer is used at 2X, not diluted. Dispense in 0.5 ml aliquots for use by the students. Store at room temperature
indefinitely.
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