Analysis of Proteins by SDS-PAGE and Western Blotting Handout

Analysis of Proteins by SDS-PAGE and Western Blotting Handout

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BIO 480W Dr. Gaillard Spring 2009 Analysis of Proteins by SDS-PAGE and Western Blotting Part I—Sample Preparation, Electrophoresis, and Blotting SDS-PAGE, short for s odium- d odecyl s ulfate p oly a crylamide g el e lectrophoresis, is a technique in which different proteins are separated from each other based on their differences in molecular weight. In SDS-PAGE, protein samples are prepared so that proteins are denatured and are surrounded by a uniform negative charge. The proteins are then loaded onto a gel matrix (Fig. 1). When an electrical current is applied, the negatively charged proteins migrate through the gel matrix toward the positive electrode in the electrophoresis chamber. Different proteins migrate through the gel at different rates, depending on their size. Larger proteins migrate more slowly through the porous gel compared to smaller proteins, which migrate relatively quickly through the gel. Thus, separation of proteins is achieved (Fig. 2). Figure 1. Cross-section through a polyacrylamide gel showing the various sizes of pores in the matrix. Figure 2. Front view of a polyacrylamide gel. The protein samples are loaded into the wells at the top of the gel. The stacking gel typically contains 4% polyacrylamide, and thus has relatively large pores. The separating gel (sometimes called the resolving gel) varies in the percentage of polyacrylamide it contains, but usually contains between 7% and 20%. The higher the percentage of polyacrylamide present, the smaller the sizes of the pores in the gel. Proteins first migrate through the stacking gel, and then enter the
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separating gel as they travel toward the positive electrode in the electrophoresis chamber. Since the stacking gel differs in pH compared to the separating gel, the proteins collapse into distinct bands as they move from the stacking gel into the separating gel. Sample Preparation The native structure of proteins is three-dimensional, and the specific shape of proteins can affect their migration through the gel. In SDS-PAGE, protein samples are prepared for electrophoresis by denaturing the proteins, so that the secondary, tertiary, and quaternary structure of proteins is removed. Ideally, the only structure left for a protein
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