Biology 225 Lab - Polyacrylamide Gel Electrophoresis of Proteins

Learning objectives By the end of this laboratory exercise you should be able to: 1. Explain the principles underlying use of electrophoresis to separate charged molecules in a mixture. 2. Explain the factors that could contribute to migration of proteins in an electric field. 3. Understand why molecular weight is the only parameter affecting protein migration in SDS-PAGE. 4. Explain why, in SDS-PAGE, all proteins migrate toward the positive pole in an electric field. 5. Describe how a protein electrophoresis procedure is carried out. 6. Explain why it is important to load several different concentrations of each tissue sample when running a polyacrylamide gel. 7. Explain how a lane consisting of molecular weight standards can be used to construct of a calibration plot for estimation of molecular weights of proteins represented by selected bands in a stained gel. 8. Understand the general relationship among cell differentiation, gene expression, and different gel band patterns associated with different tissues. Background Earlier in the course you used electrophoresis to separate DNA fragments included in a number of samples. Many electrophoretic techniques have been developed for characterization of biologically important molecules. Each technique has its own particular applications and special features, but all of the techniques share some things in common. Electrophoresis is a means of separating components in a mixture, using movement in an electric field as a basis for separation. Thus the components to be separated must be electrically charged molecules. Both proteins and nucleic acids fall into this category, and electrophoresis is most often applied to these molecules. Separation is done in some matrix, which is suspended between two electrode chambers. The matrix provides fluid-filled spaces through which charged particles can move. These spaces are filled with a buffer solution that is continuous with the buffer in the electrode chambers. Common matrices include starch, agarose, and polyacrylamide. When you used electrophoresis to separate DNA molecules earlier in the course, the separation was done using an agarose gel that you poured during the lab period. This week you will use a polyacrylamide matrix to separate protein molecules from one another. The sample is loaded into the matrix, and an electric field is imposed. Charged molecules in the sample are carried through the matrix by an electric current. Following separation, the matrix is treated in some way (e.g. it may be stained in an appropriate fashion) to reveal the locations of the molecules that have migrated from the origin. Often a standard is run along with the samples to provide a means of estimating size of the resolved fragments. When you ran the DNA gel, one well was loaded with a DNA mixture consisting of fragments of known length. The ladder that resulted was used to estimate the length (in base pairs) of fragments in the samples. Today you will load one well in your gel with a mixture of proteins of known molecular weight, and when you analyze an image of your gel you will be able to use the protein standards to estimate the molecular weights of proteins that correspond to bands on the gel. Polyacrylamide gels are porous, and the pores contribute to separation of the protein molecules. The gel can act as a sieve to retard movement of larger molecules. Thus both charge and size can contribute to the separation of molecules in a polyacrylamide gel. Because of time constraints pouring polyacrylamide gels is a procedure that requires several hours your instructors have prepared the gels you will use. As the name polyacrylamide implies, the gel forms as acrylamide monomers polymerize to form long chains. The monomer solution contains two forms of
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acrylamide; one forms short chains that that cross-links the longer chains that the other gives rise to, and the result is a three-dimensional network. The monomer solution is mixed with an appropriate buffer and a catalyst that promotes polymerization. Immediately after the mixture has been made, the liquid is poured into a mold in which the gel forms. The spaces in the three-dimensional polyacrylamide network are filled with buffer, and during electrophoresis the molecules being separated snake their way through these fluid-filled spaces. Depending on the application, the mold might be a cylindrical tube or a slab. You will be using a slab gel. The mold consists of a plain glass plate and a notched alumina plate. Two plastic spacers separate the plates. Within an hour after it has been poured into the mold, the acrylamide solution polymerizes, resulting in a sandwich: glass at one face, alumina at the other, and polyacrylamide in between. The specific recipe used for the acrylamide solution determines the pore size of the gel. A common approach employed in polyacrylamide gel electrophoresis (often referred to by the acronym PAGE) is the discontinuous system developed by Laemmli, and this is the approach you will use. The gel actually consists of two different gels, one poured atop the other. The lower gel is poured first. This gel, called the running gel or separating gel, is typically made from an acrylamide solution that is about 12% acrylamide. After the first gel has polymerized, the top gel is poured. This gel, called the stacking gel, is typically made from an acrylamide solution that is about 4% acrylamide. The stacking gel is more porous, so proteins migrate through it more quickly (and more uniformly, since a large pore gel has less of a sieving effect); as a result the proteins are condensed into a tight band before they enter the running gel. This allows for a sharper resolution of proteins within the running gel.

Figure 1: Polyacrylamide gel showing running and stacking gels (from http://www.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg) When the stacking gel was poured, a plastic comb was inserted into the sandwich before the stacking gel polymerized. When you remove the comb from your gel you will see wells left by the teeth of the comb; you will load samples in the wells.
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Several parameters contribute to the migration of protein molecules in an electric field. Small molecules move faster than do large molecules. Streamlined molecules move faster than do molecules with bulky shapes. Molecules with high charge density (charge to mass ratio) move faster than do molecules with low charge density. One common variation of PAGE is to include the detergent SDS in the buffers used to make the gel, to treat the sample, and to run the gel. In this variation (called SDS-PAGE), proteins separate on the basis of molecular weight alone. This is because the other two parameters are eliminated under the conditions used. The sample extract is heated to 95oC for 5 minutes in a treatment buffer that contains SDS. Heating break hydrogen bonds in the protein molecules, and another treatment buffer component reduces disulfide bonds (covalent bonds that contribute to protein three-dimensional structure). Thus the protein molecules lose their normal threedimensional shapes and unfold. SDS coats the unfolded protein molecules. This coating prevents the protein molecules from refolding and also masks the charges of the protein molecules. SDS itself has a negative charge, so the coated proteins will still migrate in an electric field. As a result of what happens when the proteins are treated in this fashion they all have the same streamlined shape and all have their own charges masked. With the same streamlined shape and the same charge density, the proteins differ from one another only in size, so size becomes the sole criterion for protein separation in an electric field. The treatment buffer also contains glycerol (which makes the sample dense) and a blue tracking dye (which allows you to follow the progress of electrophoresis).

Figure 2: SDS-PAGE showing effect of SDS, loading of gel, and separation of proteins by size (from http://bjpsbiotech.edublogs.org/files/2008/10/sds-page-diagram1.jpg) Protein standards of known molecular weight, treated just as the sample is treated, can be used to calibrate the gel and determine the sizes of proteins in the tissue extract. You will load one well of your gel with molecular weight standards, and the standards will be used to construct a calibration plot for the gel. This week in the laboratory you will run a polyacrylamide gel loaded with two different dilutions of three tadpole tissue extracts and with molecular weight standards. The extracts were prepared using different tissues
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 gut, liver, and muscle obtained from the same tadpoles you used for the coprophagy experiment. Earlier in the course we considered gene expression and related gene expression to presence of a functional product often a polypeptide encoded by a gene that is expressed. More recently, when we considered development, we noted that an aspect of development is cell differentiation: different specific cell types emerge, the cell types are different because of differences in patterns of gene expression, and these differences are manifested in different cell types having different protein populations. After electrophoresis, you will stain the gel with a general protein stain that binds to proteins in the gel. Because proteins in the samples separate on the basis of size, bands are established along the axis of migration in the gel. It is reasonable to expect that samples prepared from different organs will have different banding patterns. Next week in the laboratory you will make an image of your gel. As part of your report for this laboratory activity you will use the image to estimate the sizes of proteins that make up some bands in the image. Protocol 1. You will be provided previously prepared samples of tadpole tissues. Your instructors homogenized the tissues, then used a protein assay procedure to determine the protein content of each extract. The extracts were then diluted to a suitable concentration. The various gel stains commonly used for PAGE differ in their sensitivity. If too little protein is loaded, the chosen stain will be sensitive enough to allow detection of only the most prominent bands; if too much protein is loaded, too much stain will be bound, and the gel will look like a smear. Obtain a gel sandwich. Remove and discard the plastic wrap. Place the gel sandwich in the electrophoresis chamber, with the glass plate directed outward. CAUTION: Acrylamide is a neurotoxin. In the polymerized form (polyacrylamide) the substance is not hazardous. Nevertheless you must wear gloves when handling the gel, since unpolymerized monomer solution might remain on the plates or the gel surface. Discard your gloves at the end of the laboratory period. a. After you have unwrapped the gel, use a marking pen to mark the locations of the wells in the gel. On the glass plate, make a dot at the end of each tooth of the comb. Even though it is easy to see the wells when they are empty, seeing them will be much more difficult when they are filled with buffer. You must know where the wells are if you are to load the samples correctly. b. Slide the gel sandwich (comb side up) between the rubber gasket at the center of the frame for the upper gel chamber and the two side clamps. Be sure the sandwich is positioned such that the alumina plate is against the gasket of the gel chamber and the two bottom corners of the gel sandwich are resting on the orange supports at the base of the chamber. c. Position each clamp against the glass plate of the gel sandwich such that the raised lip at the lateral margin of the clamp slips past the edge of the gel sandwich. d. Carefully tighten the thumbscrews on the clamps. Turn the upper screw on one clamp just to the point at which you can feel it beginning to tighten against the gel sandwich. Then turn the lower screw on the other clamp until the clamp just begins to tighten. Repeat with the remaining two thumbscrews. Then go back through each screw to ensure it is snug. Do not over tighten, or the glass plate will break. 3. Carefully remove the comb from the top of the stacking gel by gently pulling straight up. Place the comb in the dishpan in the sink. Slip the upper buffer chamber into position in the lower buffer chamber; metal pins at the ends of the upper buffer chamber must slide into slots on extensions from the top of the upper buffer chamber. Add tank buffer to the two buffer chambers.
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b. c. d.

First fill the upper buffer chamber, and check for leaks. The upper buffer chamber is the space between the two gel sandwiches. Add buffer until the level rises above the notch in each alumina plate and fills the wells of each gel; the buffer level should be almost to the top of the glass plate on each side of the apparatus. Be sure the level of the buffer is clearly above the level of each notch. If you detect any leaks, carefully tighten the clamp thumbscrews. Once you are sure there are no leaks, fill the lower buffer chamber to the level of the yellow line found at one end of the chamber. Throughout the electrophoresis run, the lower end of the gel must be immersed in buffer in the lower buffer chamber, and the buffer in the upper buffer chamber must remain in contact with the gel over the notch in the alumina plate. If these conditions are not met the circuit will not be closed: there will be no current between the electrodes in the upper and lower buffer chambers, and the sample proteins will not move in the gel.

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Load the samples. a. The gel has 10 wells. Often migration in the outermost lanes is distorted, so you will load lanes 2-8. Load the lanes according to the following scheme. You might have to modify the loading scheme if one of wells 2-8 appears to be defective. Tissue 1 ____________________ Lane 2: 1.6 g/l solution (load 25 l) Lane 3: 1.0 g/l solution (load 25 l) Tissue 2 ____________________ Lane 4: 1.6 g/l solution (load 25 l) Lane 5: 1.0 g/l solution (load 25 l) Tissue 3____________________ Lane 6: 1.6 g/l solution (load 25 l) Lane 7: 1.0 g/l solution (load 25 l) Molecular weight standards Lane 8: load 25 l b. c. The total amount of protein in the sample lanes is 40 g (lanes 2, 4, and 6) and 25 g (lanes 3, 5, and 7). The chosen protein content for samples is within the range of total protein content (20 g to 50 g) recommended for a complex sample, such as would come from an animal organ. You must work carefully when you load the gel. 1. Be sure the electrophoresis chamber has been filled with buffer. You cannot load a dry well. During loading the sample displaces the buffer in the well. The glycerol in the treatment buffer makes the sample dense; thus when introduced into a well the sample will remain at the bottom of the well. 2. Use your P-200 with a special gel-loading tip. Because the tip has a long, skinny end you must be sure to fill it slowly, and when filling be sure to leave the tip in the sample container for several seconds after you have released the plunger. A sample of 25 l should rise about one-quarter inch into the larger diameter portion of the tip. 3. Once you have drawn up the sample, place the tip into the well, near the bottom. Be sure you do not penetrate the polyacrylamide with the tip. 4. Slowly inject the contents of the tip. You must modify your usual pipetting procedures when loading the sample. Do not pump the plunger past the first stop when loading the sample, or you will introduce air bubble and will disturb the sample. Also do not release the Pipetman plunger until you have withdrawn the tip from the well; if you release the plunger with the tip still in the well you will draw the sample back up into the tip. 5. The same tip can be used to load the two dilutions of a single tissue sample. However, remember to discard the tip whenever you change samples.
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Run the gel. a. Cover the electrophoresis chamber. The cover will fit on only one way. When the cover is in position and pressed down on the electrode posts, the red lead (positive) will be joined to the electrode in the lower buffer chamber, and the black lead (negative) will be joined to the electrode in the upper buffer chamber. Since the SDS that coats the proteins has a negative charge, all of the protein molecules are negatively charged and will migrate toward the anode in the lower buffer chamber. b. Be sure the power supply is turned off, with the voltage control knob turned all the way down. c. Attach the leads to the power supply: red to red, black to black. d. Turn on the power supply, and increase the voltage to about 200 volts; this should be near the upper end of the low range of the power supply or near the lower end of the high range on the power supply. You will notice that the voltage control knob does not allow continuous adjustment of the voltage; chose the value that is closest to 200 volts e. After the power is on, check the amperage to be sure that you have current running through the gel. If you do not have current, ask your instructor for assistance. f. Continue the run until the tracking dye approaches the bottom of the gel; notice that the bottom of the gel is actually below the surface of the buffer in the lower buffer chamber. The run will require about one hour. g. When the run is complete, turn down the voltage control knob, and turn off the power supply. Disconnect the electrophoresis chamber from the power supply. h. Remove the cover of the electrophoresis chamber. Invert the entire unit over the tray at your bench, and drain out the buffer into the tray. Pour the used buffer into the labeled flasks in the lab. Loosen the clamps, and remove your gel sandwich from the unit. Stain the gel. a. Carefully remove the gel from its mold. 1. Remember to wear disposable gloves when you are handling the gel. 2. Slip one of the spacers out of the gel sandwich, and use the spacer to pry open the sandwich from a bottom corner. If you pry from the top you may break off an ear of the notched alumina plate. The gel will usually stick to the alumina plate when you pry the plates apart. Your instructor will demonstrate how to separate the plates from one another. 3. Remove the other spacer. 4. Using a metal spatula or blade, carefully free a short stretch of the gel from the alumina plate along one side of the gel. Working over the tray at your bench, slip the nozzle tip of a wash bottle between the gel and the alumina plate. While squirting water between the gel and the alumina plate, go around the bottom and side edges of the gel (do not worry about the top of the gel), to loosen the gel from the alumina plate. 5. Carefully peel the gel from the alumina plate, and transfer the gel to a large weigh boat, which will be used as a staining dish. Be sure to write your initials and lab section on the edge of the dish so you can find your gel once it has been stained. b. Prior to staining, rinse the gel with three 5-minute deionized water washes. Cover the gel with deionized water, allow the gel to stand for 5 minutes, pour the water off, and repeat. c. After the final deionized water rinse, pour off the water, and pour on enough stain to cover the gel. d. Place the staining dish in the area designated for your lab section. Staining the gel will require several hours; your instructors will complete the gel staining and rinsing steps. e. Put the spacers and plates in the dishpan. Gel analysis. a. In the SDS-PAGE system you have used, proteins migrate as a function of the log10 of their molecular weights; the relationship between log10 MW and distance migrated is an inverse linear relationship. b. Your analysis report form includes an image of a gel and the molecular weights for the protein standards you loaded in lane 8 of your gel. Use these values, together with measurements from the gel image, to calibrate the gel. 1. Determine an Rf for each of the bands in the molecular weight standards lane.
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Measure the distance (in mm) from the stacking gel/separating gel interface to the tracking dye. This gives you the distance the tracking dye has moved through the separating gel. b. Measure the distance (in mm) from the stacking gel/separating gel interface to each band in the molecular weight standards lane. Divide each of these values by the interface-to-tracking dye distance to obtain an Rf for each protein. The values you obtain are distances the individual markers moved relative to the distance the tracking dye moved; the values will range from 0 (no migration in the separating gel) to 1.0 (movement with the tracking dye). Determine log10 MW for each of the molecular weight standards, and plot log10 MW on the vertical axis versus Rf on the horizontal axis. You should get a straight line, which represents the inverse relationship between protein electrophoretic mobility and log10 MW. If the largest and smallest molecular weight standards do not fall on what otherwise would be a good straight line, do not include these values when you make your plot. Estimate the molecular weights of chosen protein bands identified in the gel image. a. Determine the Rf for each designated band, following the procedures outlined for the molecular weight standards. b. For each band, find the calculated Rf on the horizontal axis of your plot, and draw a vertical line that intersects the calibration curve. From each point of intersection draw a horizontal line to the vertical axis of the plot. Each point of intersection is a log10 MW for the protein molecules in one of the bands. c. Determine the antilogs to obtain the molecular weights of the proteins.