How to Prepare and Run an SDS-PAGE Gel Step by Step

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for separating proteins based on their molecular weight. This method provides essential insights into protein purity, size, and quantity, making it an indispensable tool in molecular biology labs. In this blog, we will walk you through the step-by-step process of preparing and running an SDS-PAGE gel.

To begin, gather all necessary materials. You will need an electrophoresis apparatus, power supply, casting stand, gel casting plates, combs, and clips. For the gel preparation, acquire acrylamide, bis-acrylamide, Tris-HCl, SDS, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and a protein sample preparation buffer. You will also need loading dye, a protein ladder, and a staining solution such as Coomassie Brilliant Blue or a silver stain.

Start by preparing the resolving gel solution. Determine the desired concentration of acrylamide for your gel, typically between 8-15% depending on the size of the proteins to be separated. Combine acrylamide/bis-acrylamide solution, Tris-HCl buffer (pH 8.8), SDS, and distilled water. Degas the solution for a few minutes to remove air bubbles, as they can interfere with polymerization. Next, add APS and TEMED to initiate polymerization. Quickly pour the solution between the casting plates and overlay with isopropanol or distilled water to ensure a smooth surface. Allow the gel to polymerize for about 30-60 minutes.

While the resolving gel sets, prepare the stacking gel. This gel typically has a lower acrylamide concentration (4-5%) and lower pH (6.8) to facilitate protein stacking. Mix acrylamide/bis-acrylamide, Tris-HCl buffer, SDS, and distilled water, then add APS and TEMED just before pouring. Pour the solution on top of the polymerized resolving gel after removing the isopropanol or water. Insert a comb into the gel to create wells for loading samples and let it polymerize for 30-45 minutes.

Once the gel is ready, set up the electrophoresis apparatus. Carefully remove the comb and rinse the wells with running buffer to remove any unpolymerized acrylamide. Place the gel into the electrophoresis tank and fill it with SDS running buffer. Ensure that the gel is fully submerged.

Prepare your protein samples by mixing them with the loading dye, which usually contains SDS, glycerol, bromophenol blue, and a reducing agent such as dithiothreitol (DTT) or beta-mercaptoethanol. Heat the samples at 95-100°C for 5 minutes to denature the proteins and ensure they are fully coated with SDS. This will give the proteins a uniform negative charge, allowing them to be separated solely based on size.

Load the protein samples and a molecular weight marker into the wells of the gel using a micropipette. Be careful not to puncture the bottom of the wells. Attach the lid of the electrophoresis tank and connect it to the power supply. Set the voltage to 70-100 V for stacking and increase it to 100-150 V once the proteins have entered the resolving gel. Run the gel until the dye front reaches the bottom, typically taking 1-2 hours.

After electrophoresis, carefully disassemble the apparatus and remove the gel. Proceed with staining to visualize the proteins. For Coomassie staining, immerse the gel in the staining solution and gently shake for a few hours or overnight. Destain with a destaining solution until protein bands are clearly visible against a clear background. Alternatively, use a silver staining protocol for enhanced sensitivity.

Finally, analyze the results by comparing the protein bands in your sample to the molecular weight marker. Document your findings by taking photographs or scanning the gel for further analysis.

By following these steps, you should be able to successfully prepare and run an SDS-PAGE gel, allowing you to study protein samples with precision and confidence.