Protein electrophoresis Standard statement(s): 3.2.12 B Evaluate experimental information for appropriateness and adherence to relevant science processes. Interpret results of experimental research to predict new information or improve a solution. 3.3.10 B Describe and explain the chemical and structural basis of living organisms. Describe the relationship and the structure of organic molecules and the function that they serve in living organisms. 3.3.12 B Analyze the chemical and structural basis of living organisms. Identify and describe factors affecting metabolic function (e.g., temperature, acidity, hormones). 3.3.10 D Explain the mechanisms of the theory of evolution. Analyze data from fossil records, similarities in anatomy and physiology, embryological studies and DNA studies that are relevant to the theory of evolution. 3.7.10 A Identify and safely use a variety of tools, basic machines, materials and techniques to solve problems and answer questions. Select and safely use appropriate tools, materials and processes necessary to solve complex problems. 3.7.10 B Apply appropriate instruments and apparatus to examine a variety of objects and processes. Describe and use appropriate instruments to gather and analyze data. 2.5.11B Mathematical Problem Solving and Communication Use symbols, mathematical terminology, standard notation, mathematical rules, graphing and other types of mathematical representations to communicate observations, predictions, concepts, procedures, generalizations, ideas and results. Introduction and background: Gel electrophoresis is one of the most frequently used and most powerful techniques in laboratory research. In gel electrophoresis, separation of charged molecules is achieved by subjecting them to an electric current which forces them to migrate through a matrix. The behavior of molecules during gel electrophoresis depends on their sizes, shapes, and net charges. Linear DNA molecules have uniformly negatively-charged backbones and a shape that normally varies only in its length, so that migration is directly dependent on the size of the DNA fragment. With proteins, the story is different. The net charge of a protein is dependent on its amino acid content; proteins can carry a positive or a negative net charge. Similarly, the shapes of proteins vary widely. Furthermore, a protein may consist of several polypeptide subunits held together by hydrogen bonds, hydrophobic interactions, and/or disulfide bridges. Therefore, if proteins in their native configurations are electrophoresed, they will not all necessarily migrate in the same direction, and distances migrated will not be solely a function of their sizes. Thus gel electrophoresis of native proteins cannot be used to determine molecular weights of proteins, though it can provide other information on characteristics of the proteins in a mixture. To make protein migration rates a function of molecular weight, it is necessary to impose a uniform shape and charge on all the proteins in a mixture. This goal can be largely achieved by treating the protein mixture with the detergent sodium dodecyl sulfate (SDS). If a sample mixture is treated with hot SDS, the SDS disrupts all the Protein Electrophoresis Revised 05/07 1 Science In Motion Juniata College hydrogen bonds that are maintaining the protein’s three-dimensional shape. If the sample is simultaneously treated with a reducing agent such as -mercaptoethanol, disulfide bridges will also be broken, leaving the protein a linear chain of amino acids. The SDS binds to the protein backbone without regard to amino acid sequence, imparting a uniform negative charge to the molecules. Under these conditions, all the proteins in a mixture assume the same shape and charge. During electrophoresis, they all migrate toward the positive pole at a rate proportional to the log10 of their molecular weights. The focus of this lab is on separations using acrylamide. Polyacrylamide gels are composed of long linear polyacrylamide chains cross-linked with bis-acrylamide to create a network of pores interspersed between bundles of polymer. The structural features of a gel can be thought of as a three-dimensional sieve, made up of random distributions of solid material and pores. The ability of proteins or nucleic acids to move through the gel depends upon their size and structure, relative to the pores of the gel. Large molecules can usually be expected to migrate more slowly than small ones, creating separation of the distinct particles within the gel. The protein samples used in this lab will be used to demonstrate evolutionary relationships between organisms of different species. After separation of the proteins from the different species on the gels a comparison of the patterns obtained will demonstrate the relatedness of the organisms. Different species reveal variable banding patterns – “protein fingerprints”. Guiding questions: 1. What forces will cause proteins to migrate through a polyacrylamide gel? 2. Do different organisms have different proteins or the same proteins? 3. Will evolutionary relationships of organisms be evidenced through electrophoresis of proteins? 4. Why are standard curves valuable? Vocabulary: Gel Electrophoresis - Uses an electrical current to separate protein molecules of different lengths on a polyacrylamide gel. Molecules are attracted to the positive electrode. Molecules migrate at a rate inversely proportional to the size of the fragment. The larger the fragment the slower it migrates. Therefore, larger fragments do not migrate very far from the well, while smaller pieces move farther. Polyacrylamide gels – Composed of large polymers that can be thought of as a three dimensional sieve, made up of random distributions of solid materials and pores. Sodium dodecyl sulfate (SDS) – Detergent that imparts a uniform negative charge to the protein molecules. Running buffer – The tris-glycine SDS buffer provides electrical conductance and pH stabilization during the electrophoresis procedure. Protein extraction buffer - Buffer that is used to extract protein from samples and impart a negative charge to each protein molecule. Protein Electrophoresis Revised 05/07 2 Science In Motion Juniata College Kaleidoscope protein standard – Set of seven pre-stained colored proteins of known molecular weight that are used as a standard of comparison for sample proteins. Dalton – Accepted unit of measure for molecular weight. Albumin – any of numerous simple heat-coagulable water-soluble proteins that occur in blood plasma or serum, muscle, the whites of eggs, milk, and other animal substances and in many plant tissues and fluids.1 Materials: Protein size standards Pre-cast polyacrylamide gels 1X running buffer Coomassie staining solution Destain solution Staining trays (lg. weigh boats) Pellet pestles Transfer pipets Waste container Protein extraction buffer Razor blades Electrophoresis chamber and power source Boiling water bath Pipetteman and tips Protein samples Sharpies Baggies Microtubes Foam micro test tube holders (foam rack) Micro-centrifuge Scalpels Safety: Eye protection is recommended. Wear gloves when handling the gels. All reagents are non-toxic, however, the stain is very effective at staining clothing and fingers. Pre-Lab Activity: Observe the phylogenetic tree below. Of the types of fish supplied, which two do you think would be most closely related? ________________________________ and ___________________________________ Why do you feel that these fish will be the most closely related? Protein Electrophoresis Revised 05/07 3 Science In Motion Protein Electrophoresis Juniata College Revised 05/07 4 Science In Motion Juniata College Procedure: Preparation of protein extracts: 1. Label two microtubes for each fish sample being tested. Students should note that it may be helpful to label the tubes with a number code and record that code on their lab sheet. (For example: 1 & 1A = salmon) Place one set of tubes into a foam rack. 2. Using a transfer pipet2, pipet 500µl (0.5ml) of Protein Extraction Buffer into that (500µl) first set of labeled microtubes (tube 1). 3. Using the scalpels provided, cut a piece of each fish sample to approximately 0.25 X 0.25 X 0.25 cm3 . Transfer each sample into the first of its appropriately labeled microtube and cap the tube. Tubes may be kept in foam rack. 4. One by one, solubilize each tissue sample. This is accomplished by opening the tube and using a pellet pestle to grind up the fish sample. The pellet pestle must be rinsed between each fish sample to avoid cross contamination. Be sure to reclose each micro tube after grinding. 5. Incubate solubilized tissue at room temperature for 5 minutes. 6. Centrifuge the tissue sample for approx. 30 seconds in the microcentrifuge. BE SURE that the microcentrifuge is appropriately balanced. (Make and label a “blank” microtube with 500µl of water and a small chunk of protein sample, if needed, for proper balancing of centrifuge.) 7. Using a new clean transfer pipet, carefully transfer the supernatant only (no fish tissue!) into the second, appropriately labeled microtube (tube 1A). Close the cap and place the tube into the foam rack. The tube with the remaining fish tissue may now be discarded. 8. Heat the fish samples by placing the foam rack into a 95C water bath for 5 minutes. Loading and running the gels: 1. Prepare a Ready Gel cassette by cutting along the black line on the bottom of the cassette with a razor blade and pulling off the plastic strip as indicated on the gel cassette. Using a sharpie, mark the top side (short plate) of your gel cassette with your initials. Label a plastic bag with your group’s initials, the date, and your class period. 2. Remove the comb from the Ready Gel cassette. 3. Place Ready Gel cassette into the electrode assembly with the short plate facing inward. Place a buffer dam or another Ready Gel cassette on the opposite side of the electrode assembly. 4. Slide gel cassette, buffer dam, and electrode assembly into the clamping frame. Protein Electrophoresis Revised 05/07 5 Science In Motion Juniata College 5. Press down the on electrode assembly while closing the two cam levers of the clamping frame. 6. Lower the inner chamber into the mini tank3 and place your plastic bag on the lab bench in front of “your side” of the gel chamber. 7. Completely fill the inner chamber with 1x Tris-Glycine SDS Buffer, makin g sure the buffer covers the short plate (~ 150 ml). 8. Fill mini tank with approximately 200 ml of 1x buffer. 9. Place sample loading guide on top of the electrode assembly. 10. Draw a diagram of your loading pattern for your gel on your lab. 11. Load samples. Use the sample loading guide to locate the sample wells4; loading 5l of Kaleidoscope protein standard into lane one is suggested. Protein Electrophoresis Revised 05/07 6 Science In Motion Juniata College Change your pipet tip and load 5l of each sample into successive wells using a clean pipet tip for each sample. (Optional activity: Obtain other protein standards from your teacher and load them into the remaining wells.) Be sure to load samples slowly allowing them to settle evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the pipet tip. 12. Connect mini tank to power supply and set the supply to run the gel at 200 V for approximately 30 minutes5, or until dye band is about ½ inch from the bottom of the gel. Staining and destaining: 1. Once the electrophoresis is done running, disconnect electric and remove inner chamber and Ready Gel cassettes. Pour buffer back into bottle for future use. 2. Remove the top plate from the gel by gently prying it from the bottom plate. Cut the gel at the dye front by using either a spatula or a scalpel. 3. Carefully remove your gel from the bottom plate and slide it into a staining tray or ziplock plastic bag. 4. Rinse the gel 3 times for 5 minutes each time in 200 ml deionized water. 5. Once the gel has been rinsed, remove all water and add 50 ml of Bio-Safe Coomassie stain solution. Let the gel stain for 1 hour. 6. Pour the remaining stain back into the container. 7. Flood the gel with deionized water and let it sit until the bands are clearly visible. Changing the deionized water speeds up the process. Destaining overnight is most effective, but not necessary. 8. If staining procedure was performed in a staining tray, place your gel into your baggie labeled with your group’s initials, the date, and your class period after destaining is complete. View the bands on the light boxes provided. Protein Electrophoresis Revised 05/07 7 Science In Motion Juniata College Data: Make a rough sketch below of your gel. Be sure to indicate which samples were in each lane—including the Kaleidoscope standard. Note the dye front distance from the bottom of the well and distances (from the bottom of the well) of each band in cm or mm. Questions: 1. List any differences among the fish protein profiles of your samples. What is your evidence for these differences? 2. What commonalities do you see between the different protein profiles? What is your evidence for these commonalities? Protein Electrophoresis Revised 05/07 8 Science In Motion Juniata College 3. What can you conclude from these differences and similarities? Support your conclusion with evidence. 4. How do your results compare to your predictions in the pre-lab activity? If you feel that your prediction was incorrect, which species should you have chosen and why? 5. How many muscle proteins on your gel are found in both of these species of fish? 6. How many different protein bands are seen in your gel in these two species of fish? 7. To find the percentage of common proteins between these two species, perform the calculation below. (number of proteins in question #5) (number of proteins in question #6) X 100 = __________% 8. Did you observe any differences in the serum albumin samples obtained from your teacher? If so, what were the differences? 9. Referring to question #8, provide justification for why you did or did not see any differences in these serum albumin samples. Protein Electrophoresis Revised 05/07 9 Science In Motion Juniata College 10. Calculate the log10 of the molecular weights of your Kaleidoscope standard. Calculate the Rf values and graph a standard curve of log10 of MW vs. Rf. How could this standard curve be used? 11. What three physical properties influence the behavior of molecules during gel electrophoresis? 12. How were the proteins treated so that they would migrate through the polyacrylamide gel? What forces caused the proteins to migrate through that gel? References: 1 Mirriam Webster Online Database. http://www.webster.com/cgi-bin/dictionary (accessed May 24, 2007). 2,3,4,5 Bio-Rad Laboratories, Life Science Education Database. www.explorer.bio-rad.com (accessed May 24, 2007). Credits: Pictures and Products referenced produced by Bio-Rad Laboratories, Life Science Education. 1-800-4-BIORAD (800-424-6723), www.explorer.bio-rad.com--permissions for use are on file with Science In Motion. Protein Electrophoresis Revised 05/07 10 Science In Motion Juniata College Kaleidoscope Pre-stained Protein Standards Pre-stained standards consist of seven uniquely colored proteins with molecular weight range of approximately 200,000 – 6,500 daltons. Dyes have been covalently attached to the standard proteins and will not dissociate during normal staining or destaining procedures. No reconstitution or further dilution is required before use. Individual bands are easily identified by their unique colors, making it possible to monitor the separation of proteins while electrophoresis is in progress, even after the dye front has run off the gel. INSTRUCTIONS FOR USE Heat the solution to 40 for 30 seconds to dissolve any solids, which may have precipitated at -20C. To visualize the standards during electrophoresis, load 20l. To see the standards during the run, it is helpful to hold a sheet of white paper behind the gel. Protein Color Calibrated MW (Daltons) Myosin Blue 202,000 -galactosidase Magenta 121,000 Bovine serum albumin Green 79,000 Carbonic anhydrase Violet 41,000 Soybean trypsin inhibitor Orange 31,600 Lysozyme Red 17,800 Aprotinin Blue 8,200 Protein Electrophoresis Revised 05/07 11 Science In Motion Juniata College Teacher Notes For Protein Electrophoresis Lab time: This lab will take one double period or two regular class periods for protein preparation. Gel running and staining will take another double period or two more regular class periods. Target grade level: This lab is an upper-level (advanced high school) biology lab. Objectives: 1. Students will extract protein from tissue samples and migrate them through an electrophoresis gel. 2. Students will compare proteins from different organisms. 3. Students will predict evolutionary relationships of organisms and compare their predictions to electrophoresis data. 4. Students will produce a standard curve. Major concepts: Students need to understand the following information before performing the lab: The chemical structure of proteins Electrophoresis procedures How to use a pipetman—proper pipetting procedures This lab would be appropriate for a biochemical unit (after protein structure was detailed) or an evolution unit (after knowledge of phylogenic trees was discussed) or wherever deemed as appropriate for your curriculum needs. Answers to questions: Guiding questions: 1. The proteins (after being treated with the protein extraction buffer) should have the same shape and charge (a negative charge). The proteins will migrate through the gel being pulled toward the positive pole at a rate dependent upon their molecular weight. 2. Organisms possess many of the same proteins (human examples commonly known to students would be hemoglobin, insulin, etc.). Even across species, there are many common proteins. This lab demonstrates muscle proteins actin and myosin. 3. If the teacher gives a common protein—albumin (supplied by Science In Motion)—from different species to the students, students may expect that certain more related organisms will have similar albumin bands while less related organisms will have different albumin bands. The molecular weight of albumin is actually conserved across species. Only egg albumin has a vastly different molecular weight. 4. Standard curves are used to identify unknowns. Protein Electrophoresis Revised 05/07 12 Science In Motion Juniata College Pre-lab questions: Answers will vary depending upon fish samples provided. Student answers should relate to the phylogenetic tree provided. Analysis questions: Answers for questions 1-7 will vary with fish samples selected and gel electrophoresis results obtained. Teacher protein options: 8. Albumin standards should all be around the same molecular weight. Kaleidoscope standard also contains a serum albumin standard. 9. Serum albumin is highly conserved through time. Molecular weights of serum albumin molecules are fairly consistent across all species. It should be noted that serum and egg albumin are different and egg albumin molecules are different in size than serum albumin molecules. 10.Rf = distance that band travels divided by the distance the dye front travels Calibrated MW Log10 (Daltons) of MW Rf 202,000 5.30535 121,000 5.08279 79,000 4.89763 41,000 4.61278 31,600 4.49969 17,800 4.25042 8,200 3.91381 Standard curves are used in research to determine molecular weight of an unknown. Once molecular weight is known, identification (naming) of the unknown protein may be possible. 11. Sizes, shapes, and net charges all influence the behavior of molecules during gel electrophoresis. 12. The proteins (after being treated with the protein extraction buffer) should have the same shape and charge (a negative charge). The proteins will migrate through the gel being pulled toward the positive pole at a rate dependent upon their molecular weight. Protein Electrophoresis Revised 05/07 13 Science In Motion PRE-LAB Quiz POST-LAB Quiz Protein Electrophoresis Juniata College Name:_________________________ School:_________________________ Date:_________________________ 1. What forces caused the proteins to migrate through the polyacrylamide gel? A. Proteins are pulled through the polyacrylamide gel by gravity. The heavier the protein, the closer to the bottom of the gel. B. The proteins will migrate through the gel being pulled toward the positive pole at a rate dependent upon their molecular weight. C. The gel molecules will move the proteins through the polyacrylamide. D. Proteins separate out by color in the polyacrylamide gel. 2. How were the proteins treated so that they would migrate through the polyacrylamide gel? A. The proteins were treated with a protein extraction buffer that gives all of the proteins the same shape and charge. B. The proteins were treated with a polyacrylamide gel which made them equal to the electrophoresis gel. C. The proteins were not treated. There is no need to treat them prior to gel electrophoresis. D. The proteins were treated with the Kaleidoscope standard. 3. How could a standard curve of log10 of protein molecular weight vs. Rf values be used? A. These standard curves are used to create protein shapes when synthesizing proteins. B. These standard curves are used in research to determine molecular weight of an unknown. C. These standard curves cannot be used in research because of their inaccuracy. D. These standard curves are used to determine protein concentration in a gel. 4. Looking at the gel1 below, which protein seems to be conserved over time in the fish listed? Protein Electrophoresis Revised 05/07 14 Juniata College Gel Analysis Science In Motion Lane A. B. C. D. 1. Kaleidoscope Markers 2. Shark 3. Salmon 4. Trout 5. Catfish 6. Sturgeon 7. Actin and Myosin Standard Myosin light chains The Kaleidoscope protein at 41.5 Myosin heavy chains The Kaleidoscope protein at 86 5. What three physical properties influence the behavior of molecules during gel electrophoresis? a. b. c. d. Shape, color, and reflectivity Color, size, and reflectivity Size, shape, and net charge Net charge, durability, and shape 1 Bio-Rad Laboratories, Life Science Education Database. www.explorer.bio-rad.com (accessed May 24, 2007). Protein Electrophoresis Revised 05/07 15