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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