BIOTECH Project, University of Arizona
Protein Fingerprinting
Name:
Date: Period:
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:
• What characteristics are different between cows and goats? List as many as you can:
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:
1

BIOTECH Project, University of Arizona
Protein Fingerprinting
Your challenge is to design a phylogenetic tree of the organisms whose protein fingerprints you will examine.
Phylogenetic tree:
Materials/Equipment Needed
For the class:
• hot water bath (70°C)
• heat block (95°C)
• 2 thermometers
• microwave
• 3% agarose in Tris-Glycine buffer
• Tris-Glycine-SDS buffer
• Coomassie blue stain
• Destain
• Plastic wrap
• Different tissues
For each student group:
• horizontal gel electrophoresis apparatus, electrodes, power supply
• micropipet with 4 micropipet tips
• 6 microfuge tubes with 500 µl sample buffer
• 4 empty microfuge tubes
• staining tray
• paper towel or kleenex
Pouring an agarose gel:
1. Get your electrophoresis apparatus and make sure there are black stoppers at both ends of the gel tray.
2. Make sure the comb is in place at the negative electrode (black end of the gel).
3. Pour melted agarose into the gel space until the gel fills the shallow tray NOT THE WHOLE BOX.
Let the agarose harden, which should take about 10 minutes. Don’t touch/move your gel until it’s hard.
In the meantime, prepare your protein samples.
2

BIOTECH Project, University of Arizona
Protein Fingerprinting
Procedure for preparing protein samples
1. Label each of the sample buffer tubes, one for each type of tissue.
2. Cut a small bit of each tissue (size of half a pencil eraser) and put it into the corresponding tube.
3. Gently shake tubes and let samples sit for 5 minutes.
4. Label 4 empty microfuge tubes, one for each type of tissue.
5. Pipette 1/4 of the liquid (not the tissue!) from your sample tubes to the new tubes.
6. Incubate the tubes in the heat block at 95°C for 5 minutes.
7. The samples are ready to load into the gels. Be sure to keep track of which samples are loaded into which wells.
Electrophoresis
1. When the agarose gel is hard, remove the tape or stoppers.
2. Pour Tris-Glycine-SDS buffer over your gel so that is it completely covered plus a little more.
3. Draw a diagram of the gel including the wells. Label which protein samples you are putting into each well because once the samples are loaded, you will not be able to determine which sample is which.
4. Run that gel!! Plug the electrodes into your electrophoresis apparatus ( red to red, black to black ), being careful not to bump your gel too much.
5. Plug the power source into an outlet and set the voltage to 100-120 V.
6. Let the gel run until the dye migrates about 6-8 cm from the wells (about 20-30 minutes).
7. Turn off the power supply, disconnect the electrodes, and remove the top of the electrophoresis apparatus.
8. Carefully remove the gel and place it in the staining tray.
9. Pour just enough Coomassie blue stain over the gel to cover it.
10. Cover with plastic wrap and stain for at least 30 minutes. [The gels can stain for longer, but the longer they stain, the longer they will take to destain.]
11. Remove stain and pour enough destain on the gel to cover it. Tuck a paper towel or some kleenex in the holder with the gel (it will soak up the stain).
12. Cover with plastic wrap and destain overnight.
13. Put the gel holder with the gel in it on the light box and view your gel.
3

BIOTECH Project, University of Arizona
Protein Fingerprinting
Analysis
• Quickly sketch a drawing of your gel.
• Which animals' protein fingerprints look the most similar? Why?
• Based on the fingerprint similarities, which organisms do you think are most closely related?
• Which animals' protein fingerprints look the most different? Why?
• Based on the fingerprint differences, which organisms do you think are least related?
• Each band (line) in the fingerprint represents a single protein. Are there any bands in the fingerprints that look the same for all or almost all of the animals? Why might some proteins be the same in many different animals?
• Are there any bands that you can only observe in one or two animals' fingerprints? Why might some proteins not be made by all of the organisms?
4

BIOTECH Project, University of Arizona
Protein Fingerprinting
Keeping in mind what you observed from the protein fingerprints, draw a new phylogenetic tree of the organisms.
New phylogenetic tree based on protein fingerprint evidence:
Is this tree different from your initial tree? Why or why not?
Biological Classification
Until we could use DNA and protein as evidence, scientists used observable characteristics (like hair, feathers, legs, etc.) to 'classify' relationships between organisms and their ancestors. Organisms classified in the same kingdom are more closely related than those in different kingdoms. Organisms in the same phylum are more closely related that those in different phyla (plural of phylum), and so on. Conduct textbook or online research to determine the kingdoms, phyla, and classes of the organisms.
Animal Kingdom Phylum Class
According to the biological classification, which organisms are most closely related? Why?
5

BIOTECH Project, University of Arizona
Protein Fingerprinting
Which are least closely related? Why?
Does the protein fingerprinting evidence support or contradict the classifications? In other words, are the organisms more closely related according to the fingerprints, or genotype, also the organisms more closely related according to classification, or phenotype? Why do you think so?
What do you think is a more reasonable way to examine relationships between different organisms, their genotypes (evidence = protein fingerprints) or their phenotypes (evidence = observable characteristics) or both?
Why?
6