TEACHER GUIDE: Protein Fingerprinting

Big Idea:

Every cell in an organism has the recipe for every single protein in that organism's body, but what proteins does each cell actually make? During this lab investigation, students will isolate and then separate out the proteins from different bovine tissues (skeletal muscle, heart muscle, liver, etc.) using protein gel electrophoresis. This will create a 'protein fingerprint' of different types of bovine cells from a cow so students can determine whether they make the same or different proteins.

What is protein gel electrophoresis? First, proteins are what the students are going to examine with the gel. Second, the gel you will use is called agarose, a carbohydrate isolated from seaweed. Third, 'electro' means electricity. Finally, 'phorese' is a Greek word meaning 'to carry'. Students will use electricity to carry the proteins through the agarose gel. Electricity will carry the proteins because, in this experiment, the proteins are negatively charged. If the proteins were neutral (no charge), electricity would have no effect on them.

How is protein gel electrophoresis useful? It's like making spaghetti- when you finish boiling the noodles, you strain the noodles away from the water. The noodles don't go through the holes in the strainer because they're too big. If they were smaller, the noodles could fit through the holes. Proteins are like different sized noodles and the agarose gel is like a strainer, filled with little holes. Proteins that are small moves easily through the holes in the agarose gel, while larger proteins move more slowly, getting stuck in the holes in the gel. Thus, protein gel electrophoresis allows students to spread out the proteins from different bovine tissues, creating a protein fingerprint that serves as a barcode to identify that type of cell or tissue.

Almost every cell in a cow's body contains the DNA to make every protein the cow needs. The cells that don't have the complete genome (entire DNA sequence for the cow) are red blood cells, which have no nucleus, and eggs and sperm, which are haploid and each have different DNA because of recombination during meiosis. But each type of cell or tissue makes only the proteins it needs to do its job. For example, muscle cells make lots of actin and myosin, the proteins responsible for muscle contraction. One they've run their gels, students will be able to observe different protein fingerprints for each type of cell or tissue, because each cell makes a different set of proteins based on its functions.

-contains agarose powder - Tris base - glycine - distilled water

Tris-Glycine-SDS buffer (about 250 ml per group, depending on size of electrophoresis apparatus)

- contains Tris base - glycine - sodium dodecyl sulfate (SDS) - distilled water

Coomassie blue stain (about 50 ml per group, depending on size of staining trays)

- contains Coomassie blue - methanol - glacial acetic acid - distilled water

Destain (about 100 ml per group, depending on size of staining trays; contains methanol, acetic acid and distilled water)

Plastic wrap

4 different bovine tissues (possible tissues: heart, skeletal muscle [ground beef], smooth muscle [tripe], liver, kidney, thymus, testes, etc.)

For each student group (preferably groups of 4):

horizontal gel electrophoresis apparatus, electrodes, power supply
micropipet with 4 micropipet tips [or 4 transfer pipets]
4 microfuge tubes with 500 µl sample buffer (contains Tris base, sodium dodecyl sulfate, bromophenol blue and glycerol)
4 empty microfuge tubes
staining tray
paper towel or kleenex

Science Background

Sample buffer: The sample buffer contains several ingredients useful for extracting proteins and conducting gel electrophoresis. The Tris is a buffer, which helps keep pH constant during the experiment. SDS (sodium dodecyl sulfate, also known as lauryl sulfate) is a detergent that dissolves cell and nuclear membranes by breaking down lipids (fat), as well as unfolding the proteins in the sample. The bromophenol blue adds color to the solution so students can see the samples as they load them into the gel and determine when the gel has run far enough (the dye is 6-8 cm from the wells). Glycerol is a very dense liquid that makes the samples dense so they sink to the bottom of the wells in the gel.

Tris-Glycine-SDS buffer: This solution helps: keep the pH constant during gel electrophoresis (Tris); conduct electricity (glycine does this because it's charged); and keep the proteins unfolded and negatively charged so they can move through the gel toward the positive electrode (SDS does this because it is negatively charged and sticks to the proteins). Heating the proteins prior to loading them in the gel also helps unfold the proteins so they can move through the gel (Step 6 in preparing muscle samples).

Electrophoresis: Electrophoresis is an oxidation/reduction reaction. H20 splits into H+ and OH-; H+ travels to the negative electrode (black) and OH- to the positive electrode (red). At the negative electrode, H+ gains an electron (is reduced) and becomes hydrogen gas [2H+ and 2 electrons become H2 (gas)]; at the positive electrode, O2- loses two electrons (is oxidized) and becomes oxygen gas [2O2- becomes O2 (gas) and 4 electrons]. As H+ is reduced at the negative electrode, it leaves behind the base, OH-, turning the negative end basic. As O2- is oxidized at the positive electrode, it leaves behind the acid, H+, turning the positive end acidic. You'll use a buffer (Tris) to neutralize the acid and base.

Staining: While the gel is running, the students will only be able to observe the bromophenol blue. They will not be able to see any proteins (bromophenol blue doesn't stain proteins) until they stain the gels with Coomassie blue, a protein dye.

Teacher Note
This activity takes 2-3 days, depending on your class schedule. Luckily, there are several points at which the experiment can be stopped. For example, the gels can be stored at several points throughout the experiment, even over a weekend: 1. when they are poured but not yet loaded (store wrapped in refrigerator), 2. when they are run but not yet stained (store wrapped in refrigerator), and 3. when they are stained and in process of destaining (store in destain covered with plastic wrap at room temperature for up to 2 days, any longer and the proteins won't be stained enough).

Here are example timelines for conducting the protein electrophoresis experiment. You can determine what's appropriate given your schedule (whether you have a block period), your timing (do you have 2 or 3 days to do the activity?), and your supplies (1 or 2 class sets of electrophoresis equipment).

Timelines for protein gel electrophoresis

Advanced Preparation for Protein Electrophoresis Lab

1. Obtain or make all of the solutions listed in the recipes below.
   
   
2. Melt the agarose, and store melted in a 55°C water bath or on a hot plate.
   
   
3. Set up electrophoresis equipment.
   
   
4. Set heat block or second water bath to 95°C.
   
   
5. Assemble set of materials for students: 4 microfuge tubes with sample buffer, 4 empty microfuge tubes, 4 pipet tips, and one micropipet.

Pouring an agarose gel:

1. Get your electrophoresis apparatus and seal both ends of the gel tray with stoppers.
   Eventually, the students will load their protein samples in the wells at the negative end of the gel. The proteins will then migrate toward the positive end because they are negatively charged. If students accidentally plug the electrodes in backward or load into the red/positive electrode wells, their samples will migrate off the gel into the buffer instead of into the gel.
   
   
2. Make sure one comb is in place at the negative electrode (black end of the gel). Why the negative end? SDS in the sample buffer is negatively charged and sticks to the proteins in the sample, making all of the proteins negatively charged. Thus, all of the proteins placed in wells at the negative end of the gel will be attracted into the gel towards the positive electrode. While the gels are hardening, the protein samples can be prepared, if they have not been prepared already.
   
   
3. Pour melted agarose into the gel space until the gel is about 5 mm deep. Let the agarose harden, which should take about 10 minutes. Don't touch/move your gel until it's hard. In the meantime, prepare your protein samples.

Procedure for preparing muscle samples

1. Label each of the 4 sample buffer tubes, one for each type of tissue. What do you think the sample buffer does?
   The sample buffer includes bromophenol blue (allows you to see the sample while you're loading it), glycerol (which is dense and helps the samples sink to the bottom of the wells), SDS (a detergent than denatures and negatively charges proteins so they can move freely through the agarose during electrophoresis), and Tris (a pH buffer).
   
   
2. Cut a small bit of each tissue (size of half a pencil eraser) and put it into the corresponding tube.
3. Gently shake tubes and let samples sit for 5 minutes.
4. Label 4 empty microfuge tubes, one for each type of tissue.
5. Pipette 1/4 of the liquid (not the tissue!) from your sample tubes to the new tubes.
6. Incubate the tubes in the heat block at 95°C for 5 minutes. What do you think heating does?
   Heat also helps denature the proteins.
7. The samples are ready to load into the gels. Be sure to keep track of which samples are loaded into which wells.

Electrophoresis

1. When the agarose gel is hard, remove the stoppers.
2. Pour Tris-Glycine-SDS buffer over your gel so that is it completely covered plus a little more. What do you think the buffer does?Tris is a buffer that prevents changes in pH that occur during electrophoresis; SDS sticks to proteins, denaturing them and giving them a negative charge; glycine is a charged molecule that conducts electricity during electrophoresis.
3. Draw a diagram of the gel including the wells. Label which protein samples you are putting into each well because once the samples are loaded, you will not be able to determine which sample is which.
4. Run that gel!! Plug the electrodes into your electrophoresis apparatus (red to red, black to black), being careful not to bump your gel too much.You can tell the gel is plugged in and running when you see the bubbles of H2 and O2 gas forming at the electrodes. Eventually, you will see the dye in the protein samples moving toward the red/positive electrode because it too is negatively charged.
5. Plug the power source into an outlet and set the voltage to about 100 V.
6. Let the gel run until the dye migrates about 6-8 cm from the wells (about 20-30 minutes).
7. Turn off the power supply, disconnect the electrodes, and remove the top of the electrophoresis apparatus.
8. Carefully remove the gel and place it in the staining tray.
9. Pour just enough Coomassie blue stain over the gel to cover it.
10. Cover with plastic wrap and stain for at least 30 minutes. [The gels can stain for longer, but the longer they stain, the longer they will take to destain.]
11. Remove stain and pour enough destain on the gel to cover it. Tuck a paper towel or some kleenex in the holder with the gel (it will soak up the stain).
12. Cover with plastic wrap and destain overnight.
13. Put the gel holder with the gel in it on the light box and view your gel.

Analysis

1. Compare the protein fingerprints from the different tissues. What can you conclude about what proteins each type of cell makes? The protein fingerprints for each type of tissue should look different. Each type of cell makes different types of proteins, although some proteins each cell makes might be the same because some cells have to do the same jobs (e.g. cellular respiration).
   
   
2. Which tissues had the most similar proteins? Which tissues had the most different proteins? Why do you think so? The more similar the types of cells (e.g. skeletal muscle and cardiac muscle), the more similar their proteins will be. The more different the types of cells (e.g. skeletal muscle and liver), the more different the proteins in those cells will be.
   
   
3. Why would proteins from different types of cells look different? Why would they look the same? Since each cell has specific functions to perform, the more similar the cells' functions are with other cells, the more similar their proteins will be
   
   Extensions/Inquiry
   - Students can conduct a similar experiment to compare different bovine tissues to other types of meat (e.g. beef hot dogs, beef bologna, etc.).
   
   - Use this same protein electrophoresis technique to determine the evolutionary relationships between different phyla. Students could collect data by conducting a protein electrophoresis experiment with muscle tissue from different organisms (fish, fowl, mammals, invertebrates, etc.).
   
   - The Andrew Lettes extension: Students can examine differentiation (how does a stem cell, or undifferentiated cell, become a mature muscle or brain or liver cell, one that has differentiated?). Students can conduct a protein fingerprint of alfalfa seeds, one-day sprouts, and two (or more) day sprouts. They should grind up the plant tissue in a mortar and pestle with dry ice, and combining 2 small spatula scoops of the ground material with the sample buffer. Leaving the material overnight in sample buffer will allow for better protein extraction.
   
   Recipes and Background Information
   
   5X stock of Tris-Glycine buffer
   Combine 15.1 g Tris base and 94 g glycine with water to make a total volume of 1 liter. Dilute this 1:4 with water (i.e. 100 ml stock with 400 ml water to make a total 500 ml of 1X Tris-Glycine buffer). Store in a sealed bottle at room temperature indefinitely.
   
   3% agarose in Tris-Glycine buffer
   In a 250 ml Pyrex bottle, combine 3.75 g agarose with 125 ml Tris-Glycine buffer - DO NOT USE TRIS-GLYCINE-SDS BUFFER FOR THIS! You'll end up with a giant bubbly mess. Microwave uncovered for 1 minute at a time until agarose is dissolved, being careful not to let the agarose boil over on the microwave or on you! Store loosely covered at room temperature until solidified, then tightly cover to store indefinitely. Be sure to remove cover before you microwave to dissolve the agarose again.
   
   5X stock of Tris-Glycine-SDS buffer
   Combine 15.1 g Tris base, 94 g glycine, and 50 ml 10% SDS (5 g SDS with 45 ml water) with water to make a total volume of 1 liter. Dilute this 1:4 with water (i.e. 100 ml stock with 400 ml water to make a total 500 ml of Tris-Glycine-SDS buffer). Store in a sealed bottle at room temperature indefinitely.
   
   Coomassie blue stain
   For each liter of stain, combine 450 ml water, 2.5 g Coomassie blue, 450 ml methanol, and 100 ml glacial acetic. Store in a sealed bottle at room temperature indefinitely.
   
   Destain
   For each liter of destain, combine 600 ml water, 300 ml methanol, and 100 ml glacial acetic acid. Store in a sealed bottle at room temperature indefinitely.
   
   1.0 M Tris-Cl (pH 6.8)
   Combine 60.5 g Tris base with about 350 ml water. Add enough HCl to give the solution a pH of 6.8. Add enough water to make the total volume of the solution 500 ml (0.5 liter). Store in a sealed bottle at room temperature indefinitely.
   
   Sample buffer
   To make 100 ml sample buffer, combine 10 ml 1.0 M Tris-Cl (pH 6.8), 20 ml 20% SDS, 0.1 g bromophenol blue, 20 ml glycerol, and water to make a total volume of 100 ml. This is actually a 2X recipe, but the sample buffer is used at 2X, not diluted. Dispense in 0.5 ml aliquots for use by the students. Store at room temperature indefinitely.
   
   
   

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   BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona August 15, 2001 Last Modified March 6, 2002 Designed by: Nadja Anderson, Ph.D. nadja@email.arizona.edu Heather Schussman http://biotech.biology.arizona.edu