Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a widely used laboratory technique for separating proteins. This method allows researchers to differentiate proteins primarily based on their molecular weight. Understanding how to prepare the specialized gel required for this process is fundamental for effective protein separation and analysis.
Required Materials and Equipment
Preparing SDS-PAGE gels necessitates a specific collection of chemical reagents and laboratory equipment. Key chemical components include acrylamide and bis-acrylamide solutions, which form the gel matrix. Tris-HCl buffers, typically at pH 6.8 for the stacking gel and pH 8.8 for the separating gel, establish the correct pH environment for protein migration.
Sodium Dodecyl Sulfate (SDS) is also included, serving as a detergent to denature proteins and impart a uniform negative charge. Ammonium persulfate (APS) and TEMED are polymerization initiators and catalysts, crucial for gel formation. Deionized water is used for solution preparation.
Beyond chemical reagents, several pieces of equipment are essential for gel casting. This includes a gel casting stand and glass plates (one short and one long plate) to form the mold for the gel. Combs are inserted into the top of the gel to create wells for sample loading. Spatulas, measuring cylinders, pipettes, and beakers are used for mixing solutions. A magnetic stirrer and stir bar ensure homogeneous mixing, while a weighing balance is used for accurate measurement. Parafilm provides a seal.
Safety Precautions
When preparing SDS-PAGE gels, it is important to observe rigorous safety precautions due to the hazardous nature of some chemicals. Always wear appropriate personal protective equipment (PPE), including a laboratory coat, chemical-resistant gloves, and eye protection. This minimizes direct skin contact and potential exposure to chemical splashes. Working in a well-ventilated area, preferably a chemical fume hood, is advised to prevent inhalation of chemical fumes.
Acrylamide, a primary component of the gel, is a neurotoxin. TEMED is flammable and corrosive, requiring careful handling to avoid skin or eye irritation and to prevent fire hazards. Proper disposal of chemical waste according to laboratory guidelines is important after completing gel preparation.
Making the Separating Gel
The separating (or resolving) gel is the lower portion of the SDS-PAGE gel where proteins are separated primarily by size. Prepare this gel solution by combining the appropriate volumes of water, the acrylamide/bis-acrylamide solution, and the 1.5 M Tris-HCl buffer (pH 8.8). The specific percentage of acrylamide chosen for the separating gel depends on the molecular weight range of the proteins to be separated; for instance, a 12% gel is suitable for proteins between 10-70 kDa, while an 8% gel might be used for larger proteins up to 200 kDa.
Once these components are mixed, a 10% SDS solution is added, ensuring the proteins will denature and acquire a uniform negative charge during electrophoresis. The solution should be gently mixed to avoid introducing air bubbles, which can compromise gel integrity. Polymerization is initiated by adding a freshly prepared 10% ammonium persulfate (APS) solution, followed immediately by TEMED. APS generates free radicals that initiate the polymerization of acrylamide and bis-acrylamide, while TEMED catalyzes this reaction, accelerating the process. After adding APS and TEMED, the solution should be poured quickly into the gel casting plates before polymerization begins.
Making the Stacking Gel
Following the preparation and polymerization of the separating gel, the stacking gel is prepared and poured on top. The stacking gel typically has a lower acrylamide concentration, often around 4% or 5%, and a lower pH, specifically 6.8, which helps to concentrate the protein samples into a narrow band before they enter the separating gel. This concentration effect, known as “stacking,” occurs due to differences in the migration rates of ions and proteins within the gel system.
To prepare a 5 mL stacking gel solution, typical components include approximately 2.975 mL of water, 0.67 mL of 30% acrylamide/bis-acrylamide, and 1.25 mL of 0.5 M Tris-HCl (pH 6.8). Similar to the separating gel, 0.05 mL of 10% SDS is added to ensure protein denaturation. The solution is mixed gently to prevent air bubbles.
Polymerization of the stacking gel is also initiated by adding a small volume of freshly prepared 10% APS, followed by TEMED. For a 5 mL stacking gel, approximately 0.05 mL of 10% APS and 0.005 mL (5 µL) of TEMED are added just before pouring. The rapid addition of these catalysts is crucial for immediate polymerization once the solution is in the casting plates. The stacking gel’s composition ensures that proteins migrate quickly through it without significant separation, forming a tight band at the interface with the separating gel.
Pouring and Polymerizing
The physical process of pouring and polymerizing the SDS-PAGE gels requires precision to ensure a functional gel. First, clean glass plates are assembled into a casting stand, creating a sealed mold. The prepared separating gel solution is then carefully poured into the gap between the plates, typically using a pipette or serological pipette, until it reaches the desired height, leaving space for the stacking gel. It is important to pour smoothly to avoid introducing air bubbles.
Immediately after pouring the separating gel, a layer of water or isopropanol is gently overlaid on top of the gel solution. This overlay creates a flat, even surface on the separating gel as it polymerizes and also excludes oxygen, which can inhibit polymerization. The separating gel is then allowed to polymerize, a process that typically takes 30 to 60 minutes, during which the gel solidifies into a clear matrix.
Once the separating gel has fully polymerized, the water or isopropanol overlay is carefully removed, and any residual liquid is wicked away using a lint-free tissue or filter paper. The prepared stacking gel solution is then poured directly on top of the polymerized separating gel, filling the remaining space between the glass plates. A comb is immediately inserted into the unpolymerized stacking gel, ensuring no air bubbles are trapped beneath the teeth.
The stacking gel is allowed to polymerize for approximately 30 to 45 minutes. After polymerization, the comb is carefully removed, and the finished gel can be used immediately or stored at 4°C, typically wrapped in a damp paper towel and sealed in a plastic bag, for up to a few weeks.