You are using the technique of nondenaturing PAGE to separate a mixture of two pro-teins, bovine serum albumin and bovine hemoglobin. Assume that you are using a buffer of pH 8.0 and a gel of 7.5% acrylamide, and that silver staining was used for detection.
Show the results of your experiment by drawing a gel with dark bands for each protein.
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)
If protein samples are treated so that they have a uniform charge, electrophoretic mobility then depends primarily on size (see Equation 6.3). The molecular weights of proteins may be estimated if they are subjected to electrophoresis in the presence of a detergent, sodium dodecyl sulfate (SDS), and a disulfide bond reducing agent, mercaptoethanol. This method is often called denaturing electrophoresis.
The electrophoretic techniques previously discussed are called nondena-turingor “native” PAGE and are used when an investigator requires that the protein analyzed still retains its biological activity. This would be the case when the protein is an enzyme or antibody, or contains a receptor binding site. The separation of proteins under these conditions in which they maintain their native conformation is influenced by both charge and size.
When protein molecules are treated with SDS, the detergent disrupts the secondary, tertiary, and quaternary structure to produce linear polypeptide chains coated with negatively charged SDS molecules. The presence of mer-captoethanol assists in protein denaturation by reducing all disulfide bonds.
The detergent binds to hydrophobic regions of the denatured protein chain in a constant ratio of about 1.4 g of SDS per gram of protein. The bound deter-gent molecules carrying negative charges mask the native charge of the protein. In essence, polypeptide chains of a constant charge/mass ratio and uniform shape are produced. The electrophoretic mobility of the SDS-protein complexes is influenced primarily by molecular size: the larger molecules are retarded by the molecular sieving effect of the gel, and the smaller molecules have greater mobility. Empirical measurements have shown a linear relation-ship between the log molecular weight and the electrophoretic mobility (Figure 6.6).
In practice, a protein of unknown molecular weight and subunit structure is treated with 1% SDS and 0.1 M mercaptoethanol in electrophoresis buffer. A standard mixture of proteins with known molecular weights must also be sub-jected to electrophoresis under the same conditions. Two broad sets of standards are commercially available, one for low-molecular-weight proteins (molecular weight range 14,000 to 100,000) and one for high-molecular-weight proteins (45,000 to 200,000). Figure 6.7 shows a stained gel after electrophoresis of a stan-dard protein mixture. (For details on stains and staining, see Section C.) After electrophoresis and dye staining, mobilities are measured and molecular weights determined graphically.
SDS-PAGE is valuable for estimating the molecular weight of protein sub-units. This modification of gel electrophoresis finds its greatest use in character-izing the sizes and different types of subunits in oligomeric proteins. SDS-PAGE
Protein pHI MW
Bovine serum albumin 4.9 65,000 Bovine hemoglobin 6.8 65,000
0.2 0.4 0.6 0.8 1.0 1
2 Molecular weight (× 10–4)
3 4 5 6 7 8
Mobility
FIGURE 6.6 Graph illustrating the linear relationship between electrophoretic mobility of a protein and the log of its molecular weight. Thirty-seven different polypeptide chains with a molecular weight of 11,000 to 70,000 are shown. Gels were run in the presence of SDS. From K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). By permission of the copyright owner, the American Society for Biochemistry and Molecular Biology, Inc.
FIGURE 6.7 A comparison of the sensitivities achieved with three different protein stains. Identical SDS-polyacrylamide gels were stained with A SYPRO Red protein gel stain; B Silver stain; C Coomassie brilliant blue dye. Courtesy of Molecular Probes; www.probes.com, a part of Invitrogen Corporation; www.invitrogen.com.
is limited to a molecular weight range of 10,000 to 200,000. Gels of less than 2.5%
acrylamide must be used for determining molecular weights above 200,000, but these gels do not set well and are very fragile because of minimal cross-linking.
A modification using gels of agarose-acrylamide mixtures allows the measure-ment of molecular weights above 200,000.
Nucleic Acid Sequencing Gels
Sequence analysis of nucleic acids is based on the generation of sets of DNA or RNA fragments with common ends and the separation of these oligonucleotide fragments by polyacrylamide electrophoresis. Two methods have been developed for sequencing nucleic acids: (1) the partial chemical degradation method of Maxam and Gilbert, which uses four specific chemical reactions to modify bases and cleave phosphodiester bonds, and (2) the chain termination method devel-oped by Sanger, which requires a single-stranded DNA template and chain exten-sion processes, followed by chain termination caused by the presence of dideoxynucleoside triphosphates. Both sequencing methods result in nested sets of DNA or RNA fragments that have one common end and chains varying in length.
The smallest possible size difference of nucleic acid fragments is one nucleotide.
Separation of the nucleic acid fragments by polyacrylamide electrophoresis allows one to “read” the sequence of nucleotides from the gel (see Chapter 9, p. 282).
Using Sequencing Gels
The experimental arrangement is the same as that previously described for PAGE; however, the gel is prepared with many sample wells to accommodate a large number of samples. Sequence gels of 6, 8, 12, and 20% polyacrylamide are routinely used. Gels of 20% may be used to sequence the first 50 to 100 nu-cleotides of a nucleic acid, and lower percentage gels allow sequencing out to 250 nucleotides. Sequencing gels are large (up to 40 cm), and power supplies must provide more power than for conventional methods. Precast sequencing gels are now commercially available from Stratagene and other suppliers. They have a gel concentration of 5.5%, have 32 sample wells, and will sequence up to 500 nucleotides. Denaturants such as urea and formamide are required to