BIL 256 Cell and Molecular Biology Lab Spring, Molecular Weight Determination: SDS Electrophoresis

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1 BIL 256 Cell and Molecular Biology Lab Spring, 2007 Molecular Weight Determination: SDS Electrophoresis Separation of Proteins by Electrophoresis A. Separation by Charge All polypeptide chains contain at least two ionizable groups: the amino and carboxyl groups at their termini. In addition, the R-groups of most amino acid residues can be charged. These charges are responsible for the migration of proteins in an electric field. At high ph, the carboxyl (-COOH) groups are negatively charged while the amino groups (-NH 2 ) are not charged. At low ph, acidic groups are uncharged (-COOH) and basic groups (-NH 3 + ) are positively charged. Thus, there must be an intermediate ph at which the protein bears no net charge and does not migrate in an electric field. The ph at which an amino acid or protein does not migrate in an electric field is called the isoelectric point. At a ph above the isoelectric point, a protein is negatively charged and when applied to sample wells at the negative electrode end of the gel, will travel toward the positive electrode. The rate of migration of a protein species in an electric field depends upon its charge density (the ratio of charge to mass); the higher the charge density, the faster the protein will travel. For example, serum albumin, which has an isoelectric point of 4.7, will carry a strong negative charge in a buffer of ph 8.6 as compared to γ-globulin which has an isoelectric point of 7.2. Therefore, at ph 8.6 albumin will migrate toward the positive electrode at a much faster rate than γ-globulin. The agarose gel is commonly used in the charge separation of proteins since low percentage gels form a sponge-like network that serves as a medium for the buffer but which has pores large enough to allow even the largest proteins to pass unimpeded. B. Separation by Size Electrophoretic separation of proteins by molecular size requires several modifications in the basic procedure described above. First proteins are covered with the anionic detergent sodium dodecyl sulfate (SDS), which masks their native charges with its own negative charges. Proteins that contain disulfide bonds must also be treated with reducing agents to cleave these bonds so that polypeptide chains dissociate from each other and unfold. Proteins that have been treated with SDS and reducing agent assume a rod like structure and carry the same charge density imposed by anionic SDS. Since such proteins no longer possess their native shape and charge, they are referred to as denatured proteins. The final modification involves a reduction in the pore size of the agarose gel so that the gel matrix now serves as a molecular sieve. Thus, electrophoretic separation of denatured proteins sorts them according to size since it relies on the ability of uniformly charged proteins to fit through the pores of a gel matrix. Consider again the electrophoresis of albumin and γ-globulin. Albumin consists of a single polypeptide of molecular weight 66,000 Daltons (Da) while γ-globulin is a multi peptide protein containing two 53,000 Da polypeptides and two 23,000 Da polypeptides. Native γ-globulin migrates toward the positive electrode slower than albumin at ph 8.6 since it carries a weaker negative charge. When it is denatured, however, γ-globulin has the same charge density as albumin and its polypeptide chains dissociate from each other. Thus, electrophoretic migration will only be affected by the

2 sieving property of the gel matrix and the smaller, 23,000 Da γ-globulin polypeptide will move the fastest toward the positive electrode, followed by the 53,000 Da γ-globulin polypeptide and then by albumin. Polyacrylamide gels are commonly used to produce this sieving effect since small pore sizes are readily attained within the polyacrylamide gel matrix. However, the preparation of polyacrylamide gels is laborious and necessitates handling a number of toxic chemicals. Therefore, a special type of small pore size agarose gel that is comparable to the polyacrylamide gel for separating denatured proteins is used in this experiment. Agarose Gel A number of different types of stabilizing supports have been used in the electrophoretic separation of proteins. These include filter paper cellulose acetate and gels composed of either starch, polyacrylamide, agar or agarose. The agarose gel is an ideal solid support for the separation of proteins on the basis of charge, and the polyacrylamide gel is generally used for the separation of proteins on the basis of size. However, our special blend of agarose, which permits the preparation of high percentage (5-6%) agarose gels, extends the utility of this non-toxic support to SDS electrophoresis applications as well. These high percentage agarose gels have small pores, and thus, can be used to separate SDS treated proteins on the basis of size. Agarose is a natural polysaccharide of galactose and 3,6-anhydrogalactose derived from agar, which in turn is obtained from certain marine red algae. Agarose gels are made by dissolving the dry powder in boiling buffer, pouring the gels into casting trays, and allowing them to set by cooling at room temperature. Agarose gels are frequently run in the "submarine" mode where the gel is completely immersed in buffer. This feature reduces heat development in the gel that could otherwise lead to protein band distortion. Electrophoresis Buffer System The separation of proteins on the basis of molecular weight is highly dependent on their denaturation with the anionic detergent SDS. Furthermore, the ph of the buffer is also important in electrophoretic separation, since it will influence the net charge of the SDS-denatured protein. Thus, the ph and SDS concentration in the electrophoresis chamber buffer (Tris-Acetate-SDS, ph 8.3), and gel buffer (Tris-Borate, ph 8.6) that you will use in the experiment has been carefully controlled. The ionic strength of the buffer is also important in electrophoresis. High ionic strength buffers permit fast migration and can promote the sharpening of protein zones. However, high ionic strength buffers may also cause high heat production which can lead to band distortion. The moderate ionic strength buffer used in the experiment described below permits optimal resolution of protein bands in minimal time. Sample Buffer In addition to SDS denaturation, protein samples must be treated with an agent effective at eliminating disulfide bonds prior to electrophoretic separation. Thus, the sample buffer incorporates the reducing agent, β-mercaptoethanol as well as sodium dodecyl sulfate. The protein sample buffer also contains 10-20% glycerol to ensure that the samples will layer

3 smoothly at the bottom of the sample wells. The tracking dye, bromphenol blue, is also present in the sample buffer to enable the investigator to follow the progress of an electrophoretic run. Staining and Destaining Most proteins are not colored, and therefore it is necessary to visualize them in some way in order to determine their position in the agarose gel after electrophoresis. The most commonly used stain for the detection of proteins is Coomassie blue, and this stain has been incorporated into the staining solution that you will use. The staining solution also contains acetic acid which serves to precipitate and immobilize the proteins in the structure of the gel matrix after electrophoresis. The acid serves to fix the proteins in the gel so that the protein bands do not become blurred by diffusion. After the proteins in the gel have been stained, the unbound dye must be rinsed from the gel by a process known as destaining. A dilute solution of acetic acid and methanol is often used for the destaining of the agarose gels.

4 Experiment 6. Molecular Weight Determination A first step in the analysis of a protein in the molecular biology laboratory frequently involves determination of its size or molecular weight. The molecular weight of a protein may be determined using a variety of techniques. Gel electrophoresis in the presence of SDS is one of the most common methods since it is relatively simple to perform and does not require elaborate equipment. When an SDS denatured protein is separated electrophoretically, its rate of migration from the point of application toward the positively charged electrode is proportional to the log 10 of its molecular weight. When the molecular weights of several standard proteins are known, their mobilities can be plotted as a function of molecular weight to give a linear calibration curve (see Figure 1-1). The molecular weight of an unknown protein can then be estimated from this calibration curve. In practice, protein standards and unknowns are electrophoresed on adjacent lanes of the same gel. Following electrophoresis, the relative migration of standards and unknowns are determined and the molecular weight of unknowns calculated Molecular Weight Determination Rf log MW

5 In this experiment, you will determine the molecular weights of two proteins by comparing their electrophoretic migration with the migration of proteins of known molecular weight. The molecular weights of these standards are given in Table 1. You will also determine the molecular weight of the major protein that is found in rabbit serum. This protein is called serum albumin. The proteins for this experiment have been pre-stained to allow you to follow their separation during electrophoresis. Table 1-1. Molecular Weights* of Standard Proteins. Standard Protein Molecular Weight (Daltons) Bovine Serum Albumin Dimer 132,000 Bovine Serum Albumin Monomer 66,000 Ovalbumin 43,000 Myoglobin 17,000 *The terms molecular weight and Dalton are used interchangeably. For example, a 20,000 Da protein has a molecular weight of 20,000. A Dalton is a unit equal to on the atomic mass scale; this unit is very nearly equal to that of a hydrogen atom. The average amino acid residue in a protein is 120 Da. Thus, a protein with a molecular weight of 20,000 contains 167 amino acid residues (20,000/120 = 167). Gel Prep Procedure 1. Dispense 15 ml of TRIS-Borate (ph 8.6) gel buffer into a 25 ml glass test tube and then add 0.8 grams of agarose. Stir the contents of the tube with a pipet until the agarose forms a suspension. 2. Place the test tube into a boiling water bath containing a sufficient volume of water to completely immerse the agarose suspension within the tube. After 6-8 minutes in the boiling water bath, remove the test tube, stir gently, and cool at room temperature for about 2-3 minutes. At this time, the agarose solution should be clear, pale yellow, and quite viscous. 3. Insert the comb into the casting tray slots and push down gently on the top of the comb until resistance is encountered. Pour the melted agarose directly from the test tube onto the casting deck and rock the deck back and forth one time to evenly distribute the melted agarose. The teeth of the comb will come to rest in the melted agarose about 0.2 mm above the surface of the glass plate. 4. After the gel has cooled for at least 20 minutes, remove the tape strips and carefully lift the comb straight up and away from the casting tray. Cooling time can be reduced to minutes if the gel is placed in a refrigerator. The gel is now ready for sample application.

6 Sample Preparation 1. Prior to application, all samples must be vigorously boiled to encourage denaturation by SDS and β-mercaptoethanol. This is accomplished with the aid of the heat resistant foam rack. Bring to boil sufficient water to immerse but not completely cover the body of the boiling rack. Prepare the sample tubes for boiling by first tapping the tube with the tip of your index finger to mix the contents, and then piercing the top of the tube with a straight pin or similar sharp object to provide a vent for escaping steam during boiling. Once the vent has been formed, care should be taken to prevent inverting the tubes or completely submerging them during boiling. When the water is boiling vigorously, place all samples in the rack and place the rack in boiling water for a period of three minutes. 2. Load 10μl of each sample into the wells as indicated below: SAMPLE WELL SAMPLE 1,5 Standard Proteins 2,6 Transferrin 3,7 Lysozyme 4,8 Rabbit serum Pre-stained protein standards: The molecular weights of these proteins are given in Table 1-1. The cow albumin and albumin dimer are stained red-brown. The myoglobin and ovalbumin are stained blue. Transferrin: A protein sample of unknown molecular weight. The protein is stained blue. Lysozyme: A protein sample of unknown molecular weight. The protein is stained blue. Rabbit serum: The rabbit serum proteins have been pre-stained. The proteins are stained red-brown and the major protein is called albumin. This sample also contains the tracking dye, bromophenol blue. 3. Transfer the gel to the electrophoretic cell, making sure you note the positions of your samples and your gel in the electrophoresis chamber. 4. Electrophorese until the bromophenol blue in the samples has migrated to within 1 cm of the positive electrode end of the gel. 5. Remove the agarose gel from the electrophoresis unit and measure the distance each standard protein and each unknown protein has migrated in mm from the point of application (sample well). Record these values. 6. Stain and destain the gels and measure the distance of migration of each protein.