Determination of Protein Molecular Weight using SDS-PAGE
Often the first step in analyzing a polypeptide is the determination of its molecular weight or size. Molecular weights can be determined in several ways, including determination of a protein’s sedimentation coefficient. However, the majority of these techniques require the use of expensive and sophisticated pieces of equipment. Electrophoresis, on the other hand, is a relatively simple procedure to use and can be performed in any laboratory.
Electrophoresis is a technique that involves the separation of proteins or nucleic acids in an electric field. The rate of protein migration is dependent upon the net charge, size, and shape of the molecule. Electrophoresis is usually performed in gels that have formed in tubes or slabs. Tube gels are typically 12 cm long, with a 3-5 mm internal diameter, whereas slab gels are sandwiched between two pieces of glass. Both types of gels are run in the vertical position, with two buffer containers, one at the top of the gel and one at the bottom. The electrical current applied flows between the only connection between the two buffer chambers -- the gel.
In electrophoresis, current, voltage, or power is held constant throughout the gel run. There are two equations that have direct effects on the electrophoresis of proteins. One is I = E/R (current = voltage/resistance); the second is P = E x I (power = voltage x current; power is a measure of heat). The consequences of constant voltage, current, or power differ even though resistance always increases during the gel run. For example, if one holds the current (which is proportional to velocity) constant in a gel run, the resistance of the gel increases over time, and heat is generated. The heat generated must be dissipated or it will set up convections that change the pore size of the gel and hence, affect migration of proteins through the gel. Using constant voltage, the velocity slows (i.e. current drops), but little heat is generated. Therefore, it is not necessary to cool a gel during an electrophoresis run using constant voltage. Finally, if power is held constant, the velocity of migration slows, but heating is constant. Most gel systems employ either constant current or constant voltage for the electrophoresis run.
Proteins are amphoteric, meaning that they possess both positive and negative charges. The net charge of a protein is a function of the pH of the solution in which it is found. Although the amino and carboxyl groups of amino acids are responsible for most of this charge, some proteins have ionizable side groups that can also affect its net charge. If a protein is placed in a solution whose pH is above the isoelectric point (pI) of that protein, the protein will be negatively charged and migrate to the anode (electrode that attracts negatively charged molecules or anions). Likewise, if a protein is placed in a solution whose pH is below the pI, the now positively charged protein will migrate to the cathode. Moreover, all other factors being equal, the greater the difference between the pH of the solution and the pI of the protein, the farther that protein will migrate to the anode or cathode. Therefore, to achieve consistent gel runs, the pH of the buffer must be constant to prevent changes in the net charge of and mobilities of the proteins being electrophoresed. (Nucleic acids are not amphoteric, but are negatively charged at any pH routinely used for electrophoresis. Therefore, it is not necessary to prepare nucleic acids for electrophoresis.)
Usually the protein or nucleic acid is electrophoresed through a matrix such as paper, cellulose acetate, starch, agarose, or acrylamide. Regardless of the matrix, each one acts as a molecular sieve through which molecules move. Large molecules are retarded in migration through the pores, whereas smaller molecules are allowed to migrate freely. One can alter the pore size and hence alter the size of the molecular sieve. The most commonly used matrix in cell biology laboratories for protein separation is acrylamide. In making the matrix, acrylamide monomers polymerize into long chains that are linked by a crosslinker. The most common crosslinker is N,N’-methylenebisacrylamide (bis). Agarose gels can be used also, but tend to be very fragile. Moreover, the pore size created using agarose is suitable only for separation of large macromolecules, such as nucleic acids and large polypeptide complexes.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the most commonly used type of electrophoresis, separates proteins based upon molecular weight. This technique requires the proteins first be solubilized (i.e. in solution) and be devoid of secondary, tertiary, and quaternary structure. To accomplish this denaturation, proteins are treated with sodium dodecyl sulfate (SDS) and -mercaptoethanol. The SDS is an anionic detergent that binds to hydrophobic regions of proteins with its own hydrophobic hydrocarbon tail and “unfolds” the native protein. SDS also overwhelms the native charge of a protein and imparts a negative charge to all proteins regardless of intrinsic charge. -mercaptoethanol is a strong reducing agent, breaking covalent disulfide bonds between cysteine residues in polypeptides. When subjected to an electric field, these denatured polypeptides will migrate as nearly linear molecules at a rate that is proportional to the log10 of their molecular weight. Those proteins with a large molecular weight will move more slowly through the polyacrylamide than smaller proteins.
There are two SDS-PAGE systems in use today: a continuous buffer system and a discontinuous buffer system. The Laemmli (1970) system uses a discontinuous SDS system in which proteins are stacked and sorted in the stacking gel, then migrate through the resolving gel based upon size (i.e. molecular weight). This system of electrophoresis is excellent, since it gives a much better resolution of proteins than the continuous buffer system (Scopes, 1982). There are two main purposes for examining protein preparations using SDS-PAGE: to determine the molecular weight of a protein and to observe qualitative and quantitative differences among proteins from different cell preparations.
To determine the size or molecular weight of unknown proteins, a series of standards (proteins of known molecular weight) treated similarly is electrophoresed along with the unknown proteins in adjacent lanes of the gel. By measuring the distance each protein travels in mm from the origin (i.e. well), one can determine the relative mobility (Rf) of each protein. The relative mobility of a protein can be calculated using the following equation:
Rf = Distance (in mm) protein migrated
Distance (in mm) of bromophenol dye front
This value can then be plotted on semi-log paper to generate a calibration curve against which the molecular weight of unknown size proteins can be determined.
In this exercise, you will determine the molecular weights of proteins from the milk samples from last week’s lab. The standard proteins consist of the following pre-stained proteins; their colors and molecular weights are also listed:
Standard Protein Color (Daltons)
Myosin Blue 194,466
-galactosidase Magenta 127,920
Bovine serum albumin Green 87,279
Carbonic anhydrase Violet 38,848
Soybean trypsin inhibitor Orange 31,500
Lysozyme Red 17,475
Aprotinin Blue 6,814
*may not be visible on the gel
The terms dalton and molecular weight are often used interchangeably. A dalton is equivalent to the weight of a hydrogen atom and is considered equal to 1.00 on the atomic scale. The average amino acid has a mass of about 120 daltons. With this information, one can calculate the number of amino acids in a protein of known molecular weight. Likewise, given the number of amino acids that make up a protein, one can then estimate the molecular weight of a protein.
1. The SDS-PAGE gel we will use today is pre-made. The gel consists of a single dimension 0.75 mm 10% polyacrylamide gel with a stacking gel (0.5M Tris-HCl, 0.4% SDS, pH 6.8) and a resolving gel (1.5M Tris, 0.4% SDS, pH 8.8). The SDS-PAGE reducing sample buffer (blue in color) consists of 0.0625M Tris-HCl (pH 6.8), 2.0% SDS, 10% glycerol, 5% -mercaptoethanol, and 0.001% bromophenol blue. The electrophoresis tank buffer or electrode buffer in which the polymerized gel is run consists of 0.025M Tris, 0.192M glycine, 0.1% SDS.
Electrophoresis of milk samples
1. Load 20 g and 10 g (20 L and 10 L) of each of your samples and 10 L of the pre-stained Kaleidoscope standard proteins into adjacent wells on your half of the gel. Place the standards in lane 2, and the milk proteins in lanes 3-10.
2. Electrophorese the protein samples at 150 volts until the bromophenol blue marker dye has migrated to within 1 cm of the bottom of the gel. This takes about one hour.
3. Following electrophoresis, remove the gel from the electrophoresis chamber and stain overnight in Coomassie blue. The next day, examine the gel (or digital image) and record the distances (in mm) migrated by each of the standard proteins and the 5 major proteins from the milk preparations. Record your results in Table I. Record the distance traveled by the bromophenol dye front from the well to its final location on the gel.
4. Obtain a digital image of your gel. Label the imported image. Label each well with its contents and label each standard protein band with the molecular weight of that protein, not the name of the protein. Use the following figure title “Figure 1. Coomassie blue-stained SDS-PAGE gel of milk samples.”
1. Complete the table displaying the results of your calculations of the Rfvalues of proteins in the milk preparations.
2. Using Microsoft Excel, plot the Rfvalues (x axis) of the Kaleidoscope standard proteins you calculated as a function of the log of the molecular weight (y axis) as a scatter plot.
3. Using the Add Trendline option, determine the linear equation for this series of points. The equation will be in the form of y = mx+b, the general equation for a straight line (i.e. log10 of molecular weight = slope X Rf + y intercept). Also include the r2 value on the graph.
4. Print a copy of this figure and title it “Figure 2. Scatter plot of the relative mobility (Rf) as a function of molecular weight (log10) of SDS-PAGE separated Kaleidoscope standard proteins.”
5. Determine the molecular weight of the five most abundant proteins in your milk preparations. Show your work for the first molecular weight determination. Use the equation and use the Rf values you calculated for these proteins. Solving for y will give you the log10 of the molecular weight of the unknown proteins when you replace x with the Rf values you measured. To determine the molecular weight, take the antilog of the number (i.e. 10x).
Additional Questions to Answer:
1. Myosin is a major component of skeletal muscle cells. It has a molecular weight of 210,000 daltons. How far would it have migrated on your gel had it been electrophoresed with the proteins you used in this experiment? (Show your work!). What would be its Rf value?
2. The average amino acid residue has a molecular weight of 120 daltons. Assuming that your seven standard proteins contain average size amino acids, calculate the number of amino acid residues each standard protein should contain.
Would the migration of the proteins on the gel been affected if you had forgotten to add -mercaptoethanol to the samples? If so, how?
Would the migration of proteins on the gel been affected if you had forgotten to add SDS to the samples? If so, how?
4. Instead of using Coomassie blue to stain your gel, you decide to use a newly developed stain called SYPRO Ruby Protein stain, a fluorescent stain that can detect as little as 1 ng of protein. How would the banding pattern and number of bands in each well been affected had you stained the gel with SYPRO rather than Coomassie blue? If you had a protein stain with SYPRO, but not with Coomassie blue, what conclusions could you draw about the relative abundance of that protein?
5. When you compare the migration of one of your standard proteins and a protein from the milk preparation, you discover that these two proteins traveled the same distance from the sample well. What does this mean? Does it necessarily mean that these two proteins are the same? Why? Is there ever a time when you could assume they are the same protein?
What to turn in:
Table 1 (completed) + Figure 1 + Figure 2 + questions and answers
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685.
Scopes, R.K. 1982. Protein Purification: Principles and Practice. 2nd edition. Springer-Verlag, New York. 329 pp.