One widely used method for protein separation via electrophoresis employs a discontinuous polyacrylamide gel as the support medium and utilizes sodium dodecyl sulfate (SDS) to denature the proteins. This method is commonly known as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

To begin, you’ll need two gel casts, each comprising four gel plates, four gel plastics, and eight spacers. Assemble these components and secure them with the gel cast cover. Be cautious and mindful of the acrylamide kit, which should be retrieved from the refrigerator and handled in a fume hood due to potential health risks associated with excessive inhalation.

The first step involves preparing the resolving gel mix, designed for the separation of proteins based on their molecular weight. In a beaker, combine the following ingredients: 38.80 mL of nano-pure water, 19.88 mL of acrylamide, 20.00 mL of resolving buffer, 760 µL of SDS, 560 µL of APS, and 44 µL of TEMED. Carefully agitate the solution and set your pipettor to 5 mL for transferring the prepared solution (the resolving gel mix) into the four gel castings. For smooth edges, add 300 µL of isopropanol to each gel and allow them to solidify for 30 minutes.

Next, prepare the stacking gel mix, which features wells (typically 10-15 wells) to facilitate the passage of proteins. Mix 20.64 mL of nano-pure water, 3.24 mL of acrylamide, 8.04 mL of stacking gel buffer, 300 µL of SDS, 300 µL of APS, and 40 µL of TEMED. This mixture should be poured on top of the solidified resolving gel, and 10-well or 15-well combs can be used to create wells for loading your protein samples. The stacking gel is then added on top of the resolving gel.

This meticulous procedure ensures that you are well-prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a crucial technique in the realm of protein analysis.