Evolutionary Analysis of Fish Muscle Proteins
Integrated Science 4 Honors
Name _________________________Per______
Background
Can biomolecules, DNA and proteins, be used to determine similarities and differences
among species? If so, can the same comparison be used as a tool to determine the
evolutionary relationships among species? To answer these questions, we must first
consider the relationship evolution, DNA and proteins.
Evolution, change through time, occurs when the gene pool of a population changes.
These changes occur in the organisms’ DNA, resulting in changes to structure and/or
function. If these changes to structure/function– termed traits – are favorable for survival
then the organisms will reproduce and pass on those genes (and the consequent trait) to
their offspring. A simple way of remembering how biomolecules relate to one another and
to the process of natural selection is:
DNA→ RNA→ PROTEIN→ TRAIT →ADAPTATION→ SURVIVAL→ REPRODUCTION
This lab uses the technology of gel electrophoresis to make protein comparisons among
different fish species. By comparing certain proteins in different fish, we can make
predictions about how closely related they are. This may help us understand how closely
each fish species is related to the others. This information, in turn, helps us to develop
hypotheses about the evolutionary pathways that produced the species in questions.
Ultimately, these techniques may provide a better understanding of evolutionary
processes, in general, and the evolution of vertebrates, in particular.
Fish today are grouped into two major classes, Chondrichthyes (cartilaginous) and
Osteichthyes (bony) fishes. Chondrichthyes, sharks and rays, have cartilaginous rather than
bony skeletons. They do not have a swim bladder or lungs, nor do they have scales.
Osteichthyes are the most diverse class of fish. They have true scales, bony skeletons and a
swim bladder or lungs. The fish being investigated in this lab fall into these two classes,
while also including one invertebrate species for comparison.
Fish muscle tissue is used in this lab because it is low in fat and high in protein (unlike
the muscle tissue of endotherms). It is also easy to extract the protein from the muscle and
it produces clear results when run through the technology of gel electrophoresis.
Gel electrophoresis separates biomolecules by size. By placing a liquid mixture of
biomolecules (fish protein in this case) into a solid gel and exposing it to an electric current,
the different sized proteins separate and appear as different bands on the gel. These bands
can then be compared to the bands from a different fish’s proteins to determine the
similarities and differences among them. If the banding pattern between the two species is
very similar, the species are closely related. If there are many differences among the
banding patterns, then the species are more distantly related.
Pre-Lab Research/Hypotheses
Read the background handouts on ‘Proteomics’ and ‘Evolution and Classification of
Fish.’ Use this information to create a cladogram that predicts the evolutionary
relationships amongst the fish species available in this lab activity.
Procedures
Sample Preparation
1. Label a 1.5 ml flip-top microtube for each fish sample being prepared- #1-6.
2. Add 250 microliters of Bio-Rad Laemmli Sample Buffer to each labeled tube and close
the lid.
3. Cut a piece of each fish muscle (avoid skin, fat and bones) that weighs 0.15 g or has
dimensions of 0.5 x 0.5 x 1.0 cm (about the size of a Tic-Tac™) and transfer into the
appropriately labeled microtube.
4. Flick the microtube 15 times with your finger to agitate the tissue in the buffer
5. Incubate 5 minutes at room temperature to dissolve proteins.
6. Transfer the buffer containing the extracted proteins to a labeled 1.5 ml screw-cap tube
by pouring the buffer from the flip-top tube into the screw-cap tube. Note: It’s not
necessary to transfer the entire volume to the screw cap tube. Only a small volume is
actually needed for gel loading.
7. Heat the sample in the screw-cap tube for 5 minutes at 95 oC to denature proteins before
electrophoresis.
Agarose gel loading and running
1. Make sure your team’s gel box is loaded with a TGS ‘low-melt’ agarose gel and just
covered with 1x TGS buffer solution.
2. Using a fresh pipet tip, load 5 µl of the ‘kaleidoscope prestained standards’ in lane 1
of your agarose gel. Be sure to use a fresh pipet tip for each new transfer.
3. Use the table below as a guide, load 20 µl of each fish protein sample and the ‘actin
and myosin standard’ into the appropriate lane of your agarose gel.
Lane
1
2
3
4
5
6
7
8
Volume
5 µl
20 µl
20 µl
20 µl
20 µl
20 µl
20 µl
20 µl
Sample
Kaleidoscope molecular weight standard (STDS)
Fish sample 1
Fish sample 2
Fish sample 3
Fish sample 4
Fish sample 5
Fish Sample 6
Actin and Myosin standard (AM)
4. When all samples have been loaded, slide the cover of the chamber into place and
run the gels for 45 minutes at 100 Volts in 1X Tris-glycine-SDS running buffer.
Protein Fixation, Gel Staining and De-staining
1. When gels are finished running, carefully remove each gel from the gel box and
transfer into a gel staining tray.
2. Add 100 ml of gel fixative (a combination of acetic acid and ethanol alcohol). The gel
should remain in the fixative, with gentle agitation, for a minimum of 1 hour.
3. After fixing, pour off the fixative solution and add enough BioSafe Coomassie stain
to cover the gel. The gels should stain, with gentle agitation, for a minimum of 2
hours.
4. Destain the gels overnight in distilled water, with at least three changes of water.
Data Analysis
1. Visually observe the gel electrophoresis results. Which of the fish protein samples
seem to be most similar? Which are the least similar?
2. Using the light box, transparency film and a permanent marker, produce an accurate
copy of the gel results.
3. For each lane on your gel, measure the distance (in mm) that each protein band has
traveled. Carefully measure from the bottom of the well to the bottom of the protein
band. Record your results in Data Table 1, arranging the migration distances from
shortest to longest.
4. Using Data Table 1 mark an ‘X’ in each cell of the table where a specific fish has a
specific protein.
5. From Data Table 1, separately compare the number of proteins bands (X’s) Species 1
has in common with every other fish sample from the gel. Record these values in the
first row of Data Table 2. Repeat the process until each fish sample has been
individually compared to every other fish sample and the results are compiled in
Data Table 2.
6. Construct a cladogram based on the experimental results in Data Table 2. The first
branch of the cladogram (lower left) should represent the species with the fewest
shared protein bands, while the final branches (upper right) should represent the
species with the most shared protein bands. Label the branches with the letter
representing each species. Use background information on fish classification to
assign each fish type used to its correct location on the cladogram.
Analysis & Conclusions –
1. Include the drawing of your gel when you turn in this lab.
2. Include both your predicted and experimental cladograms when you turn in this lab.
3. Did your experimental results match your predicted results for the cladogram? Why or
why not?
4. Explain how the protein banding patterns relate to DNA.
5. Compare and contrast the use of proteins and DNA in this type of evolutionary
analysis. What are the relative advantages/disadvantages of each?
6. Describe at least 3 other types of evidence that could be used to test/support the
hypothesis of fish evolution suggested by your protein data from this lab activity.
Data
Data Table 1. Muscle Proteins Migration Distance
Distance
Fish Muscle Sample
Migrated
Species 1
Species 2
Species 3
Species 4
Species 5
Species 6
Data Table 2. Shared Muscle Proteins
Species 1
Species 2
Species 3
Species 6
Species 1
Species 2
Species 3
Species 4
Species 5
Species 6
Species 4
Species 5