Download simulating protein analysis using gel electrophoresis

Simulating Protein Analysis with Gel Electrophoresis – An Evolutionary Study
Integrated Science 4
Name ____________________________________ Per ______
Background
A technique known as gel electrophoresis is widely used to analyze the size of macromolecules. These
size differences can be used for evolutionary analysis as well as the analysis of a number of other critical
questions regarding both proteins and DNA. Gel electrophoresis works on two relatively simple principles.
First, when an electrical field is established with positive and negative poles, molecules are attracted to the
pole opposite their own electrical charge. It so happens that DNA and protein molecules both have an
overall negative charge and are, therefore, attracted to the positive pole in an electrical field. The second
principle involves the rate of movement in response to that charge. When moving through the same
medium as a result of the same electrical field, larger molecules move more slowly than do smaller
molecules. This means than larger molecules will travel a shorter distance over the same time period than
will smaller molecules. Consequently, when molecules of different size are exposed to the same electrical
field, for the same amount of time, they will separate according to size.
In this lab activity, you will simulate the use of gel electrophoresis in analyzing a number of muscle
proteins from vertebrate species. After that we will conduct a real laboratory activity, using gel
electrophoresis to analyze the evolutionary relationship amongst fish species based on the similarities of
their muscle proteins.
Focus Questions
• How does gel electrophoresis work?
• How can proteins be compared based on their size?
• How can comparisons of protein size be used for evolutionary analysis?
Procedure
Procedure steps #1 through #5 simply describe the steps necessary to do gel electrophoresis of vertebrate
proteins. You will need to understand those steps (and ultimately perform them in the Fish Proteins lab).
Your actual work on this lab will begin with step #6.
1. A small sample of muscle tissue is removed from each of the following species: squirrel monkey; rhesus
monkey; gorilla; lemur; human; and chimpanzee. The muscle tissue is ground up and treated with
chemicals to break open the cell membranes. Additional chemicals and heating are used to break down
the three-dimensional protein structure while leaving the primary structure of amino acids intact.
Finally more chemicals are added to stabilize the proteins in their denatured state.
2. The muscle tissue from each species is now a ‘soup’ of proteins, water and other chemicals. It contains a
number of proteins from a large number of cells. Since the cells are the same type, the proteins they
produce (and contribute to the ‘soup’) are also the same. A very small amount of this liquid contains
many millions of protein molecules.
3. A very small amount of this liquid (much less than a milliliter) is placed into a depression (called a
‘well’) at one end of a jello-like material (called an agarose gel). The gel is placed into a liquid (called a
buffer) that conducts electricity well. There is a positive electrode at one end of the gel and a negative
electrode at the other. The negative electrode is nearest to the end where the protein liquid is loaded
into the well.
4. A current of 200 volts is passed through the electrodes for (30) minutes. This current will cause the
negatively charged proteins to move through the gel towards the positively charged electrode. The
proteins will migrate at a known rate based on their size, with the smaller proteins moving a greater
distance.
5. At the end of the (30) minutes, the gels are removed from the liquid buffer. A chemical stain is added to
allow the protein molecules to be seen. They will appear as dark-colored bands at different distances
from the starting wells.
6. Use the information on protein sizes (Data Table A.) and migration rates (Data Table B.) to draw a
picture predicting the result of the gel electrophoresis described in this simulation. Use the gel map that
is provided for your drawing.
Data Table A. Muscle Protein Sizes
Species
Lemur
Protein A
1600
Muscle Protein Size (# of amino acids)
Protein B
Protein C
Protein D
Protein E
1800
3050
4100
7800
Protein F
8450
Squirrel
Monkey
Rhesus
Monkey
Gorilla
1400
1800
2300
3600
7600
8050
1010
1900
2700
3450
7200
7900
1010
1560
2850
3550
7200
8100
Chimpanzee
1150
1600
2700
3900
7250
8050
Human
1150
1560
2710
3900
6850
8050
Data Table B. Protein Migration Rates
# of amino
acids
0-1250
1260-1500
1510-1750
1760-2000
2010-2250
2260-2500
2510-2750
2760-3000
Migration
rate
(mm/30
min)
180
175
170
165
160
155
150
145
# of amino
acids
4010-4250
4260-4500
4510-4750
4760-5000
5010-5250
5260-5500
5510-5750
5760-6000
Migration
rate
(mm/30
min)
120
115
110
105
100
95
90
85
# of amino
acids
7010-7250
7260-7500
7510-7750
7760-8000
8010-8250
8260-8500
8510-8750
8760-9000
Migration
rate
(mm/30
min)
60
55
50
45
40
35
30
25
3010-3250
3260-3500
3510-3750
3760-4000
140
135
130
125
6010-6250
6260-6500
6510-6750
6760-7000
80
75
70
65
9010-9250
9260-9500
9510-9750
9760-10000
20
15
10
5
Analysis and Conclusions
1.
Construct a flow chart diagram explaining the entire procedure of gel electrophoresis as described in
Procedure steps 1-5. Be sure to include a brief explanation of why each step is necessary.
2.
From your gel map, separately compare the number of muscle protein bands the lemur has in
common with every other species’ samples on the map. Record these values in the top row of Data
Table C. below. Repeat this process until each species’ samples have been compared to every other
species and the results are compiled in Data Table C.
Data Table C. Protein Comparison Amongst Species
Squirrel Monkey
Rhesus Monkey
Gorilla
Chimpanzee
Humans
Lemur
Squirrel Monkey
Rhesus Monkey
Gorilla
Chimpanzee
3.
Construct a phylogeny based on your analysis of vertebrate muscle proteins. Briefly summarize the
results of this evolutionary analysis. Remember: the organisms with the least number of bands in
common with other organisms should be at the base of the cladogram.
8 Well Gel Map
Your Name:
Label Each Well With The Name Of The Corresponding Reaction