Gel Electrophoresis of Proteins Comparative Proteomics: Protein Profiles of Fish Muscle Tissue Adapted by Audrey Dell Hammerich, 11/12/2016 Electrophoresis is the migration of charged molecules in a strong electric field toward an electrode of the the opposite charge. This technique is widely used to examine proteins and nucleic acids. By placing the substances which are to be separated from one another in a matrix of either polyacrylamide or agrose, gel electrophoresis becomes an analytical method in which separation is achieved based upon the charge and mass of the substances. The gel matrix serves as a molecular sieve through which smaller molecules migrate more rapidly than larger molecules in the electric field. In this lab protein gel electrophoresis, the technique most widely used in biotechnology research, is used to examine muscle proteins from closely and distantly related fish species, and to identify similarities and differences in these organisms protein profiles (fingerprints). Muscle protein consists mainly of actin and myosin, but numerous other proteins also make up muscle tissue. While actin and myosin are highly conserved across all animal species, the other proteins are more diverse, varying even among closely related species. Proteomics and Evolution Proteomics is the study of proteins, particularly their structures and functions. This term was coined to make an analogy with genomics, and while it is often viewed as the "next step", proteomics is much more complicated than genomics. Most importantly, while the genome is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment. One organism will have radically different protein expression in different parts of its body, in different stages of its life cycle, and in different environmental conditions. The entirety of proteins in existence in an organism throughout its life cycle, or on a smaller scale the entirety of proteins found in a particular cell type under a particular type of stimulation, are referred to as the proteome of the organism or cell type, respectively. Variations in an organism s proteins may reflect physiological adaptations to an ecological niche and environment, but they originate as chance DNA mutations. Such random mutation events, if favorable, persist through the natural selection process and contribute to the evolution of new species with new specialized functions. The discovery of the chemical structure of DNA by Franklin, Watson, Crick, and Wilkins and the understanding of how the triplet code of nitrogen bases leads to the synthesis of proteins led to the belief that adaptations are the result of changes in the DNA code (mutations). However, current research in the field of proteomics is leading some
scientists to question whether or not DNA is the final determining factor in the synthesis of proteins and thus the determining factor in evolution. Proteomics was initially defined as the effort to catalog all the proteins expressed in all cells at all stages of development. That definition has now been expanded to include the study of protein functions, protein-protein interactions, cellular locations, expression levels, and modifications of all proteins within all cells and tissues at all stages of development. Protein Electrophoresis and SDS PAGE Polyacrylamide gel electrophoresis (PAGE) can be used to separate small molecules such as proteins. Understanding protein structure is important to understanding how PAGE can be used for protein analysis. Proteins are made of smaller units (monomers) called amino acids. There are 20 common amino acids. The sequence and interaction between these different amino acids determine the function of the protein they form. Amino acids are chemically joined together by peptide (amide) bonds to form polypeptide chains. Chains of amino acids constitute a protein. In turn these chains may interact with other polypeptides to form multi-subunit proteins. Amino acids can be combined in many different sequences. The sequence of the amino acids in the chain is referred to as the primary protein structure. Each amino acid has different properties and can interact with other amino acids in the chain. In determining the molecular weight of a protein, the secondary structure (α-helices, ß-pleated sheets), tertiary structure (e.g., protein domains held in place by disulfide bonds), and quaternary structure (e.g., several polypeptide chains folded together to form a protein) must be disrupted prior to electrophoresis. To accomplish this the proteins are treated with the detergent sodium dodecyl sulfate (SDS) and heated. SDS and heat denatures (destroys) the protein tertiary and quaternary structures, so that the proteins become less three dimensional and more linear. The dodecyl sulfate anion coats hydrophobic regions and gives the protein an overall large negative charge that is approximately proportional to the length of its polypeptide chain, allowing a mixture of proteins to be separated according to size. Heat and the Detergent SDS Denatures Proteins for SDS-PAGE Analysis 2
The proteins, in their SDS-containing Laemmli sample buffer, are separated on a gel with a matrix that acts to sieve the proteins by size upon addition of an electric current. A polyacrylamide gel is positioned in a buffer-filled chamber between two electrodes, protein samples are placed in wells at the top of the gel, and the electrodes are connected to a power supply that generates a voltage gradient across the gel. The SDS-coated, negatively charged proteins migrate through the gel away from the negatively charged anode toward the cathode, with the larger proteins moving more slowly than the smaller proteins. Protein Size Determination Biologists measure protein size in daltons (just another name for the unified atomic mass unit 1/12 the mass of 12 C), a measure of molecular mass. Most proteins have masses on the order of thousands of daltons, so the term kilodalton (kd) is often used to describe protein molecular mass. Given that the average mass of an amino acid is 110 daltons, the number of amino acids in a protein can be approximated from its molecular mass. Average amino acid = 110 daltons Approximate molecular mass of protein = number of amino acids x 110 daltons By graphing the movement of known proteins through the gel, one is able to estimate the molecular mass of the proteins (and therefore, how many amino acids they have) from the fish muscle tissue. Visualizing the Proteins Proteins in the samples are not visible while the gel is running. The only visible proteins will be those in the Precision Plus Protein Kaleidoscope standard that have been prestained with covalently attached dyes. These proteins resolve into multicolored bands that move down the gel as power is run through the gel. If the electric current is left on for too long, the proteins will run off the bottom of the gel. To guard against this and to exhibit the progress of the samples in the gel even if standards were not present, a blue tracking dye is mixed with the Laemmli sample buffer used to prepare the protein samples. This blue dye is negatively charged and is also drawn toward the positive electrode. Since the dye molecules are smaller than the proteins expected in most samples, they move ahead of the proteins in the gel. After turning off the electric current and removing the gel from the glass plates that hold it in place, the gel is placed in a stain. The stain used in this technique was originally developed to dye wool, which, like hair, is composed of protein. This stain binds specifically to proteins and not to other macromolecules such as DNA or lipids. After destaining, distinct blue bands appear on the gel, each band representing on the order of 1012 molecules of a particular protein that have migrated to that position: the larger 3
the amount of protein, the more intense the blue staining. Part I: Protein Extraction from Fish Muscle Tissue First, the proteins need to be extracted from the muscle tissue, unfolded and denatured, and each given an overall negative charge using Laemmli sample buffer, mechanical forces, and heat. In this lab small pieces of muscle tissue will be added to Laemmli sample buffer and the tissue manually disrupted by flicking the tubes. This will release muscle specific proteins from the cells, unfold them, and add an overall negative charge to each protein. The extract will then be poured off and the extracted proteins heated to 95 C, which will complete their denaturation. Extraction Procedure 1. Prepare a water bath by heating water to near boiling. The bath will be used at 95 C. 2. Label a 1.5 ml fliptop microfuge tube with the corresponding letter of each of the five fish species to be analyzed. 3. Using the provided plastic disposable serological pipets add 250 µl of Laemmli sample buffer to each fliptop tube. 4. For each sample, obtain a piece of fish muscle (avoid skin, fat, and bones) approximately 0.25 x 0.25 x 0.25 cm 3 and transfer it to the appropriately labeled fliptop tube and close the lid. 5. If fish samples are frozen thaw for at least 15 minutes. Samples must be thawed to proceed. Holding tubes in your hands can speed this process. Flick each tube 15 times to gently agitate the tissue in the buffer. Do this by holding the tube by the lid with one hand and flicking the very bottom of the tube with a single finger of the other hand. 6. Incubate the samples for 5 minutes at room temperature to extract and solubilize the proteins. While samples are incubating continue to the Gel Loading Procedure in Part 2 and prepare a Ready Gel cassette. 7. Obtain one tube containing the actin and mysin standard and one tube containing the Precision Plus Protein Kaleidoscope standard. 8. Heat the fish samples and the actin and myosin standards for 10 minutes at 95 C to denature the proteins in preparation for electrophoresis. 9. Store samples at room temperature if they are to be loaded onto the gels within 4 4
hours or store at -20 C for up to several weeks. Post Lab Questions to be answered in your lab report 1. Why did you add Laemmli sample buffer to your fish samples? 2. What was the purpose of heating the samples? 3. Have all the proteins been extracted from the fish slice or are some still left after the extraction? How could you test your hypothesis? Part II: SDS Polyacrylamide Gel Electrophoresis The proteins from fish muscle tissue have been extracted, denatured, and given a negative charge. Now they can be separated according to their molecular masses using gel electrophoresis, which will generate profiles for various fish species Preparing Gel Electrophoresis Tank Note: Wear gloves whenever contacting the gels to prevent contaminating the gels with human protein step 1 below is done for you, begin with step 2 1. Open and prepare a Ready gel cassette: a) Open the plastic bag and remove the gel. b) Discard the bag and filter paper sheets. c) Use a razor blade to slice along the dotted black line at the base of the gel. d) Pull off the clear tape strip from the bottom of the gel. Note: Make sure that the tape is removed from across the entire length of the gel bottom. There will not be a good electrophoresis and sample separation where the tape is left on. e) Remove the comb and rinse the wells using gel running buffer and a transfer pipet. 5
2. Assemble mini-protean electrophoresis module a) Place two Ready Gels facing each other (gel cassette sandwich ) into the slots at the bottom of each side of the electrode assembly with the tall plate facing outside (short plate of the gel cassette sandwich faces inward toward the notches of the U- shaped gaskets). Plates should securely fit onto the green silicone rubber gasket. This will allow the module to be filled with buffer. Note: The pictures here are for running a single gel per tank so that the back side contains a buffer dam rather than another gel. b) Open the two cam levers on the front of the clamping frame. c) Hold the gel cassettes against the assembly and slide the gel cassette sandwich and electrode assembly into the clamping frame. d) Press down on the top of the electrode assembly and close the two cams levers of the clamping frame Note: Gently pressing the top of the electrode assembly while closing the clamping frame cams forces the top of the short plate on each gel cassette sandwich to seat against the rubber gasket properly and prevents leaking e) Pour a little running buffer (an inch or so) in the upper buffer chamber to see if the assembly is leaking. If it is leaking, start the assembly over. Make sure that the gel cassettes are facing the gasket and that you are pressing down on the electrode assembly while you close the cams. 6
f) If there are no leaks, lower the electrode assembly and clamping frame (containing the two gel sandwich modules) into the gel box tank. g) Fill the inner chamber of each module center with enough running buffer (~ 125 ml) so that the level (A) reaches about halfway between the tops of the taller and shorter (B) plates of the gel cassettes. Try not to overflow the inner chamber assembly. (Dye added to make buffer visible.) h) Fill the lower buffer chamber (the mini tank) to the appropriate line on the side of the tank. Each tank should require less than 1 L of running buffer. B A Gel Loading Procedure 1. If using a sample loading guide, place it on the top of the electrode assembly between the two gels in the assembly. The guide will direct the pipet tip to the correct position for loading the sample in the well. 2. Load the gels as shown below. Note: Be sure to remove the comb from the gel cassette prior to loading, use a thin gel loading tip, and use a fresh pipet tip for each sample. 7
Well Volume Sample 1 empty 2 5 µl Kaleidoscope prestained standard 3 20 µl fish unknown 4 20 µl fish sample 1 5 20 µl fish sample 2 6 20 µl fish sample 3 7 20 µl fish sample 4 8 20 µl fish sample 5 9 10 10 µl actin and myosin standard empty 4. Load samples slowly to allow them to settle evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the pipet tip. 5. After loading all samples, remove the yellow sample loading guide (if used) and place the lid on the mini tank. Gel Electrophoresis 1. Insert the leads into the power supply, matching red to red and black to black. 2. Set the voltage to 200 V and run the gels for 30 minutes. 3. Watch for the separation of the standard. NOTE: While the gel is running, start Part III on p. 11 POWER SUPPLY DIRECTIONS C Bio-Rad PowerPac 300 Power Supply A B D 8
Bio-Rad PowerPac Basic Power Supply A C E D B To use: 1. Plug in gel box leads, matching red to red and black to black. 2. Turn power on using the switch on the right side of the unit. Note: If display flashes an error code such as E-7, turn power off and back on. The error message should clear. 3. The V light (A) should be on (the unit defaults to constant voltage). If it is not, push the Const button (B) until the V light comes on. 300 4. Enter the desired voltage by scrolling up with the arrow keys (C) on the left of the digital readout. 5. Start the power by pushing the Run (running man) button (D). 6. When the run is finished, push the Run button (D) again to stop the run. Stop button (D). The display should then read OFF Basic 4. Enter the desired voltage by scrolling up with the arrow keys (C) on the right of the digital readout. 5. Start the power by pushing the Run/Pause button (D) 6. When the run is finished, push the Stop button (E). 9
After Gel Electrophoresis Run has Finished: 7. Turn off the power supply and disconnect the leads. Remove the lid and discard the buffer from the inner chamber, release the cams, and remove the gel cassettes from the assembly. 8. Lay each cassette flat on the bench with the short plate facing up. Cut the tape along the sides of each gel cassette. Carefully pry apart the gel plates using the green metal tool provided. The gel will adhere to one of the plates. Gel Staining and Destaining Wear gloves to avoid staining your hands with the staining solution 1. Transfer the plate with the gel to a staining tray containing distilled water. The gel should detach from the plate. If not the gel may be lifted directly (and gently) from the plate and placed into the distilled water. 2. Rinse the gel 3 times with water for 5 minutes by carefully pouring out the water and replacing it. Shake it gently during the 5 minute periods. This should improve the intensity of the bands. After rinsing carefully pour off the water. 3. After rinsing place 2 gels in provided staining basins and add 50 ml Coomassie blue stain. Allow to stain for 1 hour. Gentle agitation throughout the staining time gives best results. (Better results are obtained by allowing the gel to stain until the next lab period.) NOTE: While the gel is staining, do Part III on p. 11 4. Remove from stain and place in deionized water and allow to destain overnight. 5. Alternatively gels can be destained by boiling the gel in deionized water for 3 to 6 minutes. The destained gel can be placed on Whatman paper or on white printer paper to make the bands easier to read. Post Lab Questions to be answered in your lab report 1. Why do SDS-coated proteins move when placed in an electric field? 2. What is the purpose of the actin & myosin standards and the Precision Plus Protein Kaleidoscope prestained standard? 3. Which proteins will migrate farthest? Why? 4. What is the purpose of the stain? 10
Part III: Proteomes and Molecular Evolution Calculations Molecular Weight Estimates The approximate molecular weight of a protein in a gel can be determined by comparing its migration with the migration of proteins of know size (standards). If one plots the distance traveled (in mm) for the known size proteins as a function of their molecular weight, a standard curve is generated that can be used to extrapolate the molecular weight of proteins whose size is unknown. NOTE: Semilog paper is needed since the migration of proteins through a gel is proportional to the log of their molecular weights. An extra piece is provided for analysis of your fish samples in addition to the simulated protein gel. NOTE: Start while waiting for electrophoresis of your gel. Analysis via LoggerPro Once the gel is destained it can be imagined and analyzed via the LoggerPro software. Making a Cladogram Each protein band that a fish has in common with another fish is considered a shared characteristic. A fish family evolutionary tree, or cladogram, can be constructed based on proteins bands that the fish have in common. Cladistic analysis assumes that when two organisms share a common characteristic that they also share a common ancestor with that same characteristic. In this part you will define the shared characteristics (i.e., make a list of all the different proteins in fish muscle), find which proteins are shared between fish, and construct a cladogram based on the data from a hypothetical gel. Lab Report for the simulated protein gel and your gel 1. measure and record the distances traveled for the five protein standard bands (#1 on p. 13) 2. determine the standard calibration curve by linear regression (also include the semi-log plot - #2 on p.14) 3. use the calibration curve to determine the molar masses of the proteins which migrated for each fish (include this data in a table - #3 on p.15) 4. determine which fish have which proteins in common (include this in a table - #4 on p. 15) 5. construct an evolutionary tree (cladogram) from these common proteins (#5 on p. 16) answer the 7 postlab questions above steps 1-5 are to be turned in for the simulated gel before you leave lab (group work with each student submitting his own analysis); for analysis of your gel above steps 1-3 will be done using the Logger Pro 3 software. 11
Actual Gel with standards and muscle proteins from FIVE fish species Precision Plus Protein Kaleidoscope Standards These are the standards you will be using. 12
Simulated Protein Gel for Plotting Molecular Weight Curve and Generating a cladogram Shark Salmon Trout Catfish Sturgeon mm from wells 1. To create the standard curve, measure and record the distances traveled for the five protein standard bands indicated in the simulated gel by the farthest right lane labeled Markers. Standard Band mm Traveled # of amino acids (kd/110) 37kD 25kD 20kD 15kD 10kD 13
2. On the graph paper below, plot the distances migrated in mm on the x-axis against the molecular weight of the standard bands in kd on the y-axis. Draw a line through the points. On a logarithmic scale, plotting the molecular weights against the distances migrated for each protein in the standard should result in a linear (straight line) curve. 3. Using the Simulated Fish Protein Gel on p. 13 and the standard line in the graph, complete the table shown on the following page. 14
Distance Migrated (mm) Shark Salmon Trout Catfish Sturgeon 25 X 26 X X X X 26.5 X 27.5 X X X X 29 30 30.5 32 33 34.5 35.5 36 36.5 37 37.5 38 38.5 39 39.5 40.5 41 42 44 45 46 46.5 47 47.5 51.5 52 TOTAL Molecular Weight in kd Number of Amino Acids 4. In the table enter the number of bands (proteins) pairs of fish have in common. Shark Salmon Trout Catfish Sturgeon Shark Salmon Trout Catfish Sturgeon 15
5. Now the cladogram can be constructed. First draw a line to form the trunk of your cladogram. Find the fish species with the LEAST number of bands in common with all the others. Place that species in position A below. Find the TWO species with the most bands in common and place them in positions B and C below (it doesn t matter which branch gets which fish). Identify the species with the next most bands in common to the species you placed in B and C. Place that fish in position D. The final fish species goes in position E. Your cladogram is complete. 16