Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, known as SDS-PAGE, is a widely used laboratory technique that separates proteins. This method sorts proteins based on their molecular weight. By employing an electric field and a specialized gel, SDS-PAGE analyzes complex mixtures of proteins.
The Role of Key Components
SDS-PAGE relies on its core components. A detergent, sodium dodecyl sulfate (SDS), is central to this process. SDS binds to proteins in a consistent ratio, disrupting their natural three-dimensional shape and unfolding them into a linear chain. SDS also coats the protein with a uniform negative charge, masking its intrinsic charge and ensuring separation is based solely on size.
Often, a reducing agent like beta-mercaptoethanol or dithiothreitol (DTT) is included in sample preparation. These agents break disulfide bonds, chemical links that maintain a protein’s folded structure. Breaking these bonds ensures multi-subunit proteins separate into individual polypeptide chains.
The polyacrylamide gel serves as a molecular sieve through which proteins migrate. This gel is formed from a network of cross-linked acrylamide and bis-acrylamide. The pore size of the gel can be adjusted by altering the acrylamide concentration, to separate proteins within specific size ranges. Higher acrylamide concentrations result in smaller pores, better for resolving smaller proteins, while lower concentrations create larger pores suitable for larger proteins.
The electrophoresis buffer fills the chamber and maintains stable pH and electrical conductivity. A power supply provides the electric field, driving the negatively charged, SDS-coated proteins from the negative electrode (cathode) towards the positive electrode (anode).
The Electrophoresis Procedure
SDS-PAGE involves preparing protein samples. Proteins are mixed with a sample buffer containing SDS and often a reducing agent, then heated. This denatures the proteins into linear, negatively charged molecules. The sample buffer also contains glycerol, which increases density, helping samples sink into gel wells, and a tracking dye, such as bromophenol blue, to visually monitor migration.
The polyacrylamide gel is often cast in two layers: a stacking gel and a separating (or resolving) gel. The stacking gel, at the top, has a lower acrylamide concentration and pH, which concentrates proteins into a narrow band before they enter the separating gel. The separating gel, with its higher acrylamide concentration and different pH, is where size-based separation occurs.
Once the gel is prepared, treated protein samples are loaded into individual wells at the top of the stacking gel. A molecular weight ladder, consisting of proteins with known molecular weights, is loaded into one well as a reference. After loading, the gel is placed into an electrophoresis chamber filled with running buffer, and an electric current is applied.
The negatively charged proteins migrate through the porous gel matrix towards the positive electrode. Smaller proteins encounter less resistance and move faster, traveling further down the gel. Larger proteins face more resistance and migrate more slowly, remaining higher up.
After the run, typically when the tracking dye reaches the bottom, the proteins are still invisible. To visualize them, the gel is removed and stained, most commonly with Coomassie Brilliant Blue. This dye binds to the proteins, making them appear as distinct blue bands against a clear background after destaining. Other sensitive staining methods like silver staining or fluorescent dyes can also be used.
Interpreting the Results
The stained gel provides information about proteins. Each distinct band represents a different protein or protein subunit. The position of these bands is directly related to the protein’s molecular weight. By comparing the migration distance of unknown protein bands to the molecular weight ladder, researchers can estimate their proteins’ molecular weight. This estimation is typically done by creating a standard curve, plotting the migration distance of known markers against their logarithmic molecular weights.
SDS-PAGE also reveals protein purity and relative quantity within a sample. A purified protein sample shows a single, prominent band at its expected molecular weight. Multiple bands may indicate a mixture of proteins or degradation products. The intensity of a band provides a relative measure of the protein’s abundance, allowing for comparisons across different samples.
While powerful for size-based separation, SDS-PAGE has specific characteristics. The technique separates proteins based on their polypeptide chain length after denaturation, not their native structure or biological function. Additionally, certain post-translational modifications, such as glycosylation, can affect a protein’s migration, potentially causing it to appear at an apparent molecular weight different from its calculated mass. Despite these considerations, SDS-PAGE remains a widely used method for protein analysis.