Electrophoresis: Tips, tools, protocols, and theories

Electrophoresis is a daily operation in many labs. Have you thought about how good is you gel electrophoresis? Is the protocol of traditional gel electrophoresis you are using a good one? Think again now, you will see how much you can change for your daily gel electrophoresis experiments.

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The irresistible beauty of novel electrophoresis from Biokeystone

Electrophoresis: Protein SDS PAGE

The solution of proteins for electrophoresis is first mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in folding, as differences in folding patterns would cause some proteins to better fit through the gel matrix than others. Adding SDS solves this problem, as it linearizes the proteins so that they may be separated in electrophoresis strictly by length (primary structure, or number of amino acids). The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass: charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. A tracking dye may be added to the protein solution to allow the user to track the progress of the protein solution through the gel during the electrophoresis run.

SDS-PAGE electrophoresis

Besides the addition of SDS, proteins may optionally be boiled in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-Mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE, and is most commonly used electrophoresis. Non-reducing SDS-PAGE electrophoresis (no boiling and no reducing agent) may be used when native structure is important in further analysis (e.g. enzyme activity, shown by the use of zymograms).

Electrophoresis and Staining

The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer. During electrophoresis an electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores of the gel, while larger ones will have more difficulty. After electrophoresis, the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Thus proteins may be separated roughly according to size (and therefore, molecular weight). Following electrophoresis, the gel may be stained (most commonly with Coomassie Brilliant Blue or silver stain), allowing visualization of the separated proteins, or processed further (e.g. Western blot). After staining, different proteins will appear as distinct bands within the gel. It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.

Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size. False negatives and positives are possible. A co-migrating contaminant can appear as the same band as the desired protein. This co-migration in electrophoresis could also cause a protein to run at a different position or to not be able to penetrate the gel. This is why it is important to stain the entire gel including the stacking section. Coomassie blue will also bind with less affinity to glycoproteins and fibrous proteins, which interferes with quantification (Deutscher 1990).
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Buffer Systems in electrophoresis

Most protein separations are performed using a "discontinuous" buffer system that significantly enhances the sharpness of the bands within the gel after electrophoresis. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. Negative ions from the buffer in the tank then "outrun" the SDS-covered protein "stack" and eliminate the ion gradient so that the proteins subsequently separate by the sieving action in the lower, "resolving" region of the gel.

Many people continue to use a tris-glycine or "Laemmli" buffering system for their electrophoresis that stacks and resolves at a pH of ~8.3-9.0. These pHs promote disulfide bond formation between cysteine residues in the proteins, especially when they are present at high concentrations because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-Tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pHs is that the acrylamide gel is more stable so the gels can be stored for long periods of time before use.

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