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Determining Evolutionary Relationships between Eight Different Fish Species by Comparison of
Muscles Proteins by Electrophoresis
Theodora Triphon
University of the Pacific
March 22, 2011
2 Blue
Dr. Lisa Wrischnik
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Abstract: In this experiment, proteins from the muscle tissue of eight fish species was
extracted and underwent SDS-polyacrylamide gel electrophoresis to separate proteins based on
their size. The proteins were then stained and the resulting banding patterns were analyzed to
see the similarities in concentration and protein size. The species analyzed were cod, salmon,
halibut, catfish, shark, calamari, swordfish, and shrimp. Species with the most similar banding
patterns and protein weights are most closely related. The hypothesis is that swordfish is more
related to halibut than the other six species analyzed. If halibut’s and swordfish’s protein
banding patterns are the most similar, then they are the most related and share a most
common recent ancestor. Using a kaleidoscope standard, an equation between the distances
proteins traveled across the polyacrylamide gel and the weight of the specific proteins was
obtained. The equation was used to find the weights of proteins of various species, and the
similarities and differences of the protein fingerprints were used to analyze the evolutionary
relatedness of the species. It was found that swordfish and halibut were closely related, as they
shared several protein bands and the darkness of these bands were similar, proving they had
similar or identical proteins in relatively equal concentrations. From these results, swordfish
and halibut are more closely related to each other than most of the other species analyzed.
Introduction: While there are millions of diverse species, all organisms share a common
ancestor. Because of this ancestry, all life on Earth is related, as all organisms have DNA and
similar mechanisms for metabolism and reproduction. Studies have shown that a great deal of
DNA sequence similarity exists among the genes of all modern-day organisms. Since DNA
provides instructions for protein making, this means that many of the proteins in organisms are
similar or identical. Mutations of the DNA will cause different proteins to be made, so different
species have different proteins or different concentrations of proteins. Therefore, it can be
concluded that organisms that are most closely related would have similar proteins and
quantities of proteins in specific tissue types.
Proteins make up the phenotype of an organism, and are also enzymes that interact in
various chemical reactions in all over the body. Proteins also provide structure, especially in
muscle tissue. The two major proteins that make up muscle fibers are actin and myosin, and are
ubiquitous in all animals (Bio-Rad). These are proteins that are identical in several species.
Molecular systematics such as protein comparison can be more evolutionarily
informative than classifying organisms based on their morphology (Campbell and Reece).
Proteins from different species can be compared, though the individuals may have different
proteins as they have all evolved separately from a common ancestor. The similarities between
the proteomes of the different organisms can be analyzed to see how related certain species
are (Bio-Rad) Protein size and quantity of each protein present in a muscle sample from a
particular animal, gel electrophoresis is used. Electrophoresis is a technique that separates
molecules based upon charge, size and shape. It can be used to create protein band patterns of
the organisms, and they can be compared to see which species are more related than others.
The proteins are denatured and given a negative charge by the detergent SDS, and are pulled
across polyacrylamide gel by a positive charge. The heaviest proteins will move slowly across
the gel due to its size, and the smaller proteins will move farther across the gel. The gel is then
stained so that specific types of proteins that have been separated and compared.
Electrophoresis of muscle proteins and the protein banding patterns formed was used to
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evaluate the question of how evolutionarily related are shark, shrimp, calamari, swordfish, cod,
salmon, catfish, and halibut. After observing the phylogenetic tree in the Bio-Rad Protein
Fingerprinting lab, the hypothesis is that swordfish is more related to halibut over the other six
species analyzed. If the protein fingerprint of swordfish is most similar to the protein fingerprint
of halibut, then the two species are closely related and share a recent common ancestor. The
experiment comes from a modified Bio-Rad 2002 Protein Fingerprinting: Comparison of Fish
Muscle Proteins by Electrophoresis.
Methods and Materials: High-resolution polyacrylamide gel electrophoresis was used to
compare the proteins in the muscle tissues of the different fish. First, the proteins were
extracted from the sample tissues of each organism. Eight flip-top microtubes were labeled the
names of the fish samples to be analyzed. 250 𝝁L of Laemmli Sample Buffer was added to each
labeled tube in order to give each protein a negative charge. A small piece, around one gram, of
each muscle sample from each organism was added to each labeled microtube. The microtube
was flicked fifteen times to fully expose the tissue to the sample buffer. The samples were
incubated at room temperature for five minutes at room temperature in order to extract the
proteins. The solutions from each microtube were poured into screwcap tubes, and then they,
along with an actin and myosin standard, were heated at 95°C for five minutes to denature the
proteins in order for them to be used in electrophoresis. After the proteins had been extracted,
given a negative charge, and denatured, they were ready to be placed in the gel wells. The
comb and tape along the bottom of the Ready Gel cassette were removed, and it was placed on
one side of the electrode assembly and a buffer dam on the other side. The Ready gel cassette
was sealed in place.
Figure 1: Order of samples in the Ready
The space between the two gel cassettes was filled
Gel cassette.
with buffer so that the inner short plates were covered.
Lane Volume Sample
Buffer was also added into the lower buffer chamber. A
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10𝝁L
Kaleidoscope
yellow sample loading guide was placed on top of the
prestained
electrode assembly, and helped direct the pipette tip to
standard
the correct position for loading each sample in a well. Each
2
10𝝁L
Halibut
sample was assigned to a well, and standards were placed
3
10𝝁L
Cod
on the ends of the wells. The order of the samples in the
Ready Gel cassette is shown in Figure 1. 10 𝝁L of each
4
10𝝁L
Salmon
protein sample was added to its designated gel well. After
5
10𝝁L
Catfish
removing the sample loading guide, placing the lid on the
6
10𝝁L
Shark
tank, inserting the leads into the power supply, the gels
were run at 250 V for twenty minutes.
7
10𝝁L
Calamari
When the gels were finished running, the power supply
8
10𝝁L
Shrimp
was turned off and the electrode assembly and clamping
9
10𝝁L
Swordfish
frame were removed. The buffer was poured out, and the
gel cassettes were removed. Gloves were used when
10
10𝝁L
Actin/Myosin
handling the gel to avoid contamination, and the tape was
Standard
cut along the sides of the gel cassette. The gel plates were
carefully pried apart and the gel was removed then added to Silver Blue stain for five minutes.
Also, a fixation step was added, where a fixing solution of 10% acetic acid, 50% ethanol, and
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40% water acetic acid and ethanol was added to the gel so that the proteins would bind in the
gel. The gel was removed and ready for analysis.
Results:
Figure 2: 15% polyacrylamide Ready Gel electrophoresed at 250V for 20 minutes, stained
with Blue Silver stain and destained in water.
The purpose of this lab was to determine the relationships between the proteins of the eight
fish through analysis of protein banding. Figure 2 shows the banding patterns of each organism
as well as the kaleidoscope standards and the actin/myosin standard. If in each organism’s
fingerprint there is the same protein, they all have that protein. Darkness of stains indicates the
concentrations of the various proteins, and organisms with a thick dark stain have a high
concentration of a certain protein. This was done to see how evolutionarily related the
organisms are. The kaleidoscope standard is helpful because its proteins have a known mass, so
after measuring each protein’s migration, it is possible to make a graph. This graph is shown in
Figure 3. The R² value proves that the correlation between migration and protein size is
positive. From the equation of the graph, any protein’s mass can be determined by measuring
its migration across the gel. Once the migration was found, it was inputted into the equation,
which then gave the mass of each protein. Figures 4 and 5 shows the various proteins of each
species and their migrations. Figure 6 shows the proteins which each species shares with all the
others. Halibut and Swordfish shared four proteins, whereas they each shared less with the
other species analyzed.
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Mass of protein (kD)
1000
Distance of Migration v. Mass of protein
y = 365.34e-0.052x
R² = 0.9126
100
Series3
10
y = 252.27e-0.045x
R² = 0.8688
Kaleidoscope
Standard
Actin Myosin
Standard
1
0
50
100
Distance Protein Migrated (mm)
Distinct bands of the fingerprints of each species was measured and through the equation
formulated
from of
theKaleidoscope,
kaleidoscopeActin
protein
theStandards
mass of each
wasof
found,
as shown
Figure 3: Mass
andgraph,
Myosin
(kD) protein
v. Distance
Migration
of
in Figures 4 and 5. The similarities of theStandards
bands were
counted.
As
seen
in
Figure
6,
halibut
and
(mm)
swordfish shared four protein bands.
Halibut
mm kD
Cod
mm kD
Salmon
mm kD
Catfish
mm kD
43
28.39696
42
29.26177417
42
29.26177
42.5
28.82612
46
25.95287
46
25.95286762
49
23.71914
46
25.95287
49
23.71914
49
23.71913651
52
21.67766
49
23.71914
51
22.33784
53
21.03698861
63
15.58459
52
21.67766
53
21.03699
59
17.57157205
76
10.55165
56.5
18.94012
56
19.22636
63
15.58458761
0
103.16
64
15.12399
61
16.54828
23
0
103.16
77
10.23981
76
10.55165
51.7426
Figure 4: Mass and distance traveled by individual proteins of halibut, cod, salmon, and catfish.
Shark
Squid
Shrimp
Swordfish
mm kD
mm kD
mm kD
mm kD
41
30.15293
43
28.39696
42.5
28.82612
42
29.26177
46
25.95287
49
23.71914
47
25.18584
48
24.44149
52
21.67766
52
21.67766
50
23.01813
53
21.03699
58
18.10671
57
18.65814
55
19.81189
63
15.58459
60
17.05225
63.5
15.35256
64
15.12399
67
13.82229
63
15.58459
66
14.24324
67
13.82229
71.5
12.07676
65
14.67701
75
10.873
103.16
23
51.7426
67.5
13.6165
77.5
10.08736
103.16
0
Figure 5: Mass and distance traveled by individual proteins of Shark, Squid, Shrimp, and
Swordfish
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Cod Salmon Catfish Shark Squid Shrimp Swordfish Halibut
Cod
5
2
1
2
2
0
3
3
Salmon
2
7
2
2
2
0
0
2
Catfish
1
2
8
1
2
0
1
3
Shark
2
2
1
9
1
1
1
2
Squid
2
2
2
1
8
0
1
1
Shrimp
0
0
0
1
0
6
1
0
Swordfish 4
0
1
1
1
1
6
4
Halibut
4
2
3
2
1
0
4
Figure 6: Number of Band Proteins shared between Species
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Discussion and Conclusion: Electrophoresis of muscle proteins was used to answer the
question of how evolutionarily related are shark, shrimp, calamari, swordfish, cod, salmon,
catfish, and halibut. The hypothesis was that swordfish is more related to halibut over the other
six species analyzed. If the protein fingerprint of swordfish is most similar to the protein
fingerprint of halibut, than the two species are closely related and share a recent common
ancestor. I conclude from the figures above that halibut is the closest relative of swordfish
among the sample organisms. This assumption is made upon first inspecting figure 2. The
fingerprints of swordfish and halibut are very similar, with only a few exclusive proteins of each
species. Also, the darkness of each band differs because each species may have different
concentrations of the same proteins, but both species have relatively similar band prominence.
The darkness of the individual bands is very similar, such as the similar color of the myosin
heavy chains. However, halibut has a darker stain for actin, proving maybe that it is different
than halibut because actin is more important to its function. After finding the mass of several
proteins of each species, the numbers of band proteins shared between species were listed in
figure five to see if halibut and swordfish were the closest related to each other. The results in
figure five shows that halibut shared four proteins with swordfish, proving that they are more
closely related than halibut is with other fish. This proves that swordfish and halibut most likely
share a common ancestor.
The actin/myosin standard was used to test the accuracy of the bandings. The bandings did not
match the true values, so this could cause errors with other readings between closely related
species. Errors during the experiment could have included improper staining of the gels, or not
complete fixation of proteins to the gel. The gel is distorted on the right side, and this could
lead to complicated measurement. Errors during the results could have been from improper
measurement of the migration of kaleidoscope proteins, which would cause the entire graph
and equation to be incorrect. This error could lead to incorrect measurement of protein mass.
Also, incorrect measurements of protein migrations of the different species could cause many
errors. It would cause an incorrect measurement of kilodaltons, and it could cause errors in
comparing proteins, as some may be matching but incorrectly measured so they cannot be
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acknowledged as the same protein. A way to avoid some of these errors would be to run
several identical gels, and see if there is a flaw with the materials or method.
For the future, this sort of experiment can be used to validate existing phylogenetic trees, as
other organisms relationships’ can be tested in the same way. This type of experiment can be
extended beyond fish, and even to humans for medical research, as well as studying hominids
and how closely related their proteins were to ours.
In conclusion, examination of the darkness of bands, the overall appearance of the protein
fingerprints, and calculating the mass of various proteins proves that halibut and swordfish are
the most closely related to each other among the eight species in the experiment.
References:
Bio-Rad. “Comparison of Fish Muscle Proteins by Electrophoresis” in Protein Fingerprinting.
2002. pp.1-11
Campbell, Neil A., and Jane B. Reece. "Chapter 25 Phylogeny and Systematics." Biology. 8th ed.
Print.
"Fish Protein Fingerprinting on Polyacrylamide Gel." Web. 14 Mar. 2011.
<http://www.carolina.com/text/teacherresources/instructions/biotech/fish_protein_fingerp
rinting.pdf>.