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MARIETTA BIOL 309 - Electrophoresis and Electroblotting of Proteins

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SDS-PAGE & Electroblotting of Proteins Page 1 Figure 1. Movement of negatively charged proteins in an electric field. Electrophoresis and Electroblotting of Proteins The purpose of the next lab exercises will be to study the relative amounts of β-actin in cells of the B-16 melanoma, liver and muscle of mice. Electrophoresis is used to separate proteins in a mixture and study certain properties, such as molecular weight, subunit structure, and expression level. Electrophoresis is frequently coupled with ‘immunoblotting’, in which antibodies are used to analyze even the rarest (least abundant) proteins in a tissue. Linking electrophoresis and immunoblotting is a methodology called ‘electroblotting’, used to prepare proteins separated by electrophoresis for immunoblotting. This week, you will perform the electrophoresis and electroblotting protocols. The principles of Electroblotting are described later in this lab exercise, and the principles of Immunoblotting are described in the next chapter. After reading the background information, complete all prelab questions and calculations in your lab notebook. Objectives The objectives of this lab exercise are for you to: - learn the theory, practice, and applications of gel electrophoresis, electroblotting and immunoblotting - understand how gel electrophoresis can be used to determine the molecular weight of proteins. - Study the levels of actin in different tissues and compare actin levels in melanoma vs normal tissues. How does gel electrophoresis work? "Electrophoresis" refers to the use of an electric field to separate proteins in a mixture. Most proteins have a negative ionic charge due to a preponderance of acidic amino acids on the surface. Thus, when placed in an electric field most proteins move toward the positive electrode through electrical attraction (Figure 1). In “gel electrophoresis”, electrophoresis is performed through a porous, semirigid matrix (or "gel"). Because different types of protein have unique amino acid composition, they have differences in ionic charge which cause the proteins to move toward the anode at different speeds. After they are separated, the proteins will remain immobilized within the gel in their final positions. Several different types of materials, including starch, agarose and polyacrylamide can be used to form the gel. Each material has its own unique properties and special applications. The material most widely employed for separation of proteins is polyacrylamide. Polyacrylamide gels are perfectly clear, and this facilitates analysis of the results. After the electrophoresis is complete, the positions of the proteins within the gel can be revealed by staining.SDS-PAGE & Electroblotting of Proteins Page 2 Figure 2. Formation of a polyacrylamide gel. Figure 3. Appearance of gel after the electrophoresis is complete and protein bands have been stained. What is a polyacrylamide gel? Polyacrylamide is formed through the polymerization of acrylamide monomer subunits (Figure 2). Acrylamide monomers are polymerized by adding ammonium persulfate and TEMED (tetramethylenediamine), which produce free-radicals that catalyze the polymerization reaction. Acrylamide monomers polymerize into long linear chains. Also included with the acrylamide is a "crosslinker" molecule called bis-acrylamide, which cross-links the polyacrylamide molecules to form a semirigid, porous matrix though which the proteins migrate. Along with the features of polyacrylamide described above, the concentrations of acrylamide and bis-acrylamide can be varied to yield a matrix with a porosity best suited to a particular application. One disadvantage of polyacrylamide gels is that the unpolymerized acrylamide monomer is a neurotoxin, and must be carefully handled. In the polymerized form, however, polyacrylamide is not toxic. When performing gel electrophoresis, a Tracking dye, such as bromphenol blue, is added to the samples. This small, negatively charged molecule is used to monitor progress of the electrophoresis. The electrophoresis is stopped when the tracking dye reaches the bottom edge of the gel. What factors influence the rate at which proteins move through a polyacrylamide gel? At the end of the electrophoresis, the final positions of the protein are visualized by staining of the gel. A typical result is shown in Figure 3. Different types of proteins form bands as they move through the gel. Why do proteins move through the gel at different rates? The relative rate of movement depends upon the charge, molecular weight, and shape of the protein. Proteins with a greater negative charge are attracted more strongly toward the positive electrode, and therefore move faster. The molecular weight and shape of the proteins are factors because of the properties of the gel matrix. The gel matrix imposes a "sieving" effect upon the movement of proteins; the larger the protein the slower its movement. But shape also is important: other factors being equal, small globular proteins move more rapidly than large elongated proteins.SDS-PAGE & Electroblotting of Proteins Page 3 y = -1.0769x + 5.30700.10.20.30.40.50.60.70.80.944.2 4.4 4.6 4.85Protein mass (log MW)Protein mobility (Rf)Figure 4. Relationship between protein MW and mobility in SDS-PAGE. Stacking Gel: 4% Acrylamide, pH 6.8 Resolving Gel: 12% acrylamide, pH 8.8 Figure 5. Arrangement of stacking and resolving gels in a disc gel system. How can protein MW be determined using “SDS - polyacrylamide gel electrophoresis” (SDS-PAGE)? The type of electrophoresis described above is called native protein electrophoresis because the proteins retain their natural (native) 3-dimensional structure as they move through the gel. This type of electrophoresis is very useful for studying the properties of proteins in their natural form. However, generally, native proteins do not separate very well during gel electrophoresis. Furthermore, native gel electrophoresis cannot be used to determine the molecular weight of proteins, because, as we described above, movement through the gel is also influenced by the shape and charge of the protein. However, the molecular weight of proteins can be determined using a procedure known as sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For SDS-PAGE, the protein sample is first treated with SDS, an ionic detergent which causes the proteins to unfold, or


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