Download Ichthyology Proteomics Profile

Ichthyology Proteomics Profile
(the study of fish)
(the study of proteins)
Protein Synthesis Review: The central dogma of DNA is diagrammed quite simply.
The flow sheet shows the relationship between DNA, mRNA, tRNA and the
polypeptide that is produced in protein synthesis. The monomers of proteins are
amino acids and there are 20 in Nature. If similar cells of 2 different species are
extracted, gel electrophoresis technology can be used to separate the different proteins
within those cells. If the gel reveals the cells have a completely different set of
proteins, it is likely that those 2species are not closely related. However, protein
similarity between 2 species would indicate they share an evolution history; the more
proteins they have in common the more similar their evolutionary pathway. In using
proteomics (study of the structure and function of proteins) science is able to use
proteins to compare species’ relatedness and build cladograms to show evolutionary
progression. Proteins are also used as an ‘evolutionary clock’ which can indicate how
long ago a change occurred between different species.
Place the terms below on the
Central Dogma of DNA flow sheet
Terms
A. Anti-codon
B. Codon
C. DNA Polymerase
D. Helicase
E. Nucleus
F. Polypeptide
G. Ribosome
H. RNA Polymerase
The ‘Old School’ Ichthyology Study
(the study of fish)
Instructions: Look at the enlarged color pictures of the 5 fish, catfish, cod, salmon,
perch and tilapia. Using their morphology, determine which fish are closely related
and which fish are not closely related. Draw a phylogenetic tree for the 5 fish and
label the ‘tree branches’. Refer to Phylogenetic Tree activity if you have forgotten
Phylogenetic Tree of 5 Fish
(Based on external morphology & label the ‘tree branches’)
Showing evolution relationships using external body characteristics of fish is an ‘old
school’ (get the pun?) approach and has been replaced by gathering molecular
information (DNA, proteins, amino acid sequence) of the species and presenting that
information as a cladogram instead of a phylogenetic tree. In this dry lab, we will be
using electrophoretic gels to compare the proteins of these same 5 fish and you will
create a cladogram from the protein data that gathered from the electrophoretic gel.
PREPARING THE PROTEIN SO THAT IT MAY RUN
THROUGH THE GEL
Proteins are very large, globular (blobby) macromolecules. We need to prepare the
protein so that it will actually travel through the gel which has very small holes in it.
The gel acts as a sieve once the electricity is turned on. We must take the protein from
its ‘blobby’ stage down to its simplest form, an amino acid chain. This requires
denaturing the protein from its globular form but we must keep the amino acids chained
together.
Diagram A
1. What are 3 different items / techniques that can be used to denature an enzyme?
(hint: think back to the liver lab 1st semester, ahhh, good times)
A.
B.
C.
A hot water bath and a buffer (that will adjust the pH and give the protein a slight
negative charge) are used to prepare the globular protein for the gel.
Once the protein has been properly prepared, it is loaded into the wells of the gel with a
micropipette
Diagram C
Diagram B
The completed gel is below BUT you will use a color photo of the fish gel to get the
data. The color photo is in a plastic sleeve with a completed Proteins Standard Graph
on the flip slide of the sleeve. You will need to read the PSG in order to calculate the
molecular weight of each fish band of the blue gel
1. Measure how far the first protein band traveled for the Catfish. Use the Protein
Standard Graph to approximate the weight of each fish protein band.
2. Record your data in Data Chart 1 of the lab
3. Continue until all 5 Catfish bands have been measured and the protein weight
approximated. Complete reading the gel for all the fish and their proteins
Data Chart 1
Distance
Protein Band
Migrated
(mm)
7
7.5
8
8.5
9
9.5
10
10.5
11
11.5
12
14
14.5
15
15.5
16
16.5
17
18
27
30
34
34.5
37
37.5
41
41.5
46
47.5
48
48.5
51
53.5
Total Protein
Bands per Fish
Protein Bands Approximate Molecular Weight
Catfish
(kD)
Cod
(kD)
Data Chart 1
Protein Bands Approximate Molecular Weight
Salmon
(kD)
Perch
(kD)
Tilapia
(kD)
Instructions: Write the number of how many proteins each fish pairing has in
common. Use Data Chart 1 as your reference source to fill in Table 2
Table 2
Number of Proteins 2 Fish Have in Common
Catfish Cod Salmon Perch Tilapia
Catfish
Cod
Salmon
Perch
Tilapia
From your Proteomics Data, create a Cladogram of the 5 fish. Remember, the
outgroup is the most ancestral fish (no derived character), the other 4 fish are ingroups
because they have at least 1 derived character. As you progress up the cladogram
‘ladder’, each ingroup has more of the derived characters. Label the derived character
(protein information) on the ‘ladder. Refer to Create your Own Cladogram activity if
you have forgotten
Cladogram of 5 Fish
(Based on proteomic gel information)
Questions:
1. According to your Phylogenetic Tree …….
A. Which 2 fish are most closely related?
B. Which 2 fish are least closely related?
2. According to your Cladogram …..
A. Which 2 fish are most closely related?
B. Which 2 fish are least closely related?
3. Between the 2 evolution timelines, (phylogenetic tree / cladogram) which is
probably more accurate? EXPLAIN your answer
You measured the distance each fish protein band migrated from the well and
using the PSG as a baseline, approximated each band’s molecular weight.
4. What is the relationship between distance migrated and molecular weight of the
fish bands?
5. Where have you dealt with this relationship before? (hint: Who’s Your Dolphin
Daddy?)
When you extracted DNA from a strawberry, you had to add a buffer (liquid
aspirin) to your slurry. A buffer is also used when running a protein gel
6. What are 2 possible functions of buffers ( 1 for DNA extraction & 1 for
proteomics)
A.
B.
In the Strawberry DNA Extraction Lab, we had to denature the histones of DNA
7. Why did the histones have to be denatured?
8. What item did we use to denature the histones?
9. How were the globular fish proteins denatured in this paper proteomics lab?
Summary Paragraph Write a paragraph using the terms, UNDERLINE the terms in
the paragraph. If
I can not read your answer, then it is wrong
Terms
Central Dogma of DNA
Evolutionary relationships
Morphology
Phenotype
Proteins