TEACHER GUIDE: Protein Fingerprinting
BIG IDEA: Every cell in an organism has the recipe for every single protein in that organism's body, but what
proteins does each cell actually make? During this lab investigation, students will isolate and then separate out
the proteins from different bovine tissues (skeletal muscle, heart muscle, liver, etc.) using protein gel
electrophoresis. This will create a 'protein fingerprint' of different types of bovine cells from a cow so students
can determine whether they make the same or different proteins.
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 in a cow's body contains the DNA to make every protein the cow needs. The cells that don't
have the complete genome (entire DNA sequence for the cow) are red blood cells, which have no nucleus, and
eggs and sperm, which are haploid and each have different DNA because of recombination during meiosis. But
each type of cell or tissue makes only the proteins it needs to do its job. For example, muscle cells make lots of
actin and myosin, the proteins responsible for muscle contraction. Liver cells make LDL receptor protein in
order to take up cholesterol from the bloodstream. One they've run their gels, students will be able to observe
different protein fingerprints for each type of cell or tissue, because each cell makes a different set of proteins
based on its functions.
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
• 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
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• Plastic wrap
• 4 different bovine tissues
(possible tissues: heart, skeletal muscle [ground beef], smooth muscle [tripe], liver, kidney, thymus,
testes, etc.)
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
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
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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)
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)
Advance Preparation for Protein Electrophoresis Lab
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.
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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
are negatively charged because SDS, which is negatively charged, is in the gel buffer and sticks to proteins,
making them negative as well.]
Extensions/Inquiry
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• Students can conduct a similar experiment to compare different bovine tissues to other types of meat (e.g. beef
hot dogs, beef bologna, etc.).
• Use this same protein electrophoresis technique to determine the evolutionary relationships between different
phyla. Students could collect data by conducting a protein electrophoresis experiment with muscle tissue from
different organisms (fish, fowl, mammals, invertebrates, etc.).
• The Andrew Lettes extension: Students can examine differentiation (how does a stem cell, or undifferentiated
cell, become a mature muscle or brain or liver cell, one that has differentiated?). Students can conduct a protein
fingerprint of alfalfa seeds, one-day sprouts, and two (or more) day sprouts. They should grind up the plant
tissue in a mortar and pestle with dry ice, and combining 2 small spatula scoops of the ground material with the
sample buffer. Leaving the material overnight in sample buffer will allow for better protein extraction.
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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