SDS-PAGE also called sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique used to separate proteins only by their size. SDS or sodium dodecyl sulfate is a detergent used to denature the proteins; they will unfold and will not have any secondary, tertiary or quaternary structure. Proteins will have a negative charge and they will move down the gel by their molecular weight. PAGE or polyacrylamide gel electrophoresis is a polymer of the neurotoxin acrylamide and a cross-linking agent called bis-acrylamide. Usually APS and TEMED are used as catalysts for the process of cross-linking. There are two different settings of running the polyacrylamide gel, denaturing gel and non-denaturing gel or native gel. Denaturing gel consists of using urea or SDS to denature or unfold proteins and subsequently separate them by their molecular weight. A non-denaturing or native gel is used primarily to analyze the tertiary and quaternary structure of proteins; therefore they do not need to be unfolded. SDS-PAGE is essential in protein analysis in different fields such as forensic, biochemistry, biotechnology, molecular biology and genetics.

The SDS-PAGE gel has to be poured in two parts. The first gel that is poured is the resolving gel. This type of gel consists of 3 to 30 % acrylamide thus possessing small pores and a pH of 8.8. The second gel is called the stacking gel and it is poured on top of the resolving gel. Stacking gel has 8 to 12 % acrylamide, therefore possessing large pores and a pH of 6.8. The main purpose of the stacking gel is to aid all the proteins to enter the resolving gel simultaneously. SDS-PAGE uses a loading buffer that contains glycerol, beta mercaptoethanol and a tracking dye. Glycerol and beta mercaptoethanol are responsible for breaking the disulfide bonds, while a tracking dye, for example bromophenol blue, is used for observing the sample migration. Meanwhile the running buffer contains glycine ions. These ions are zwitterion meaning that they have two different states depending on the pH. The stacking buffer contains chloride ions that will move faster towards the positive electrode. In the stacking gel, the glycine ions will lose their charged due to the low pH and will slow down, while the chloride ions will move faster. Next, an electric current is applied causing the proteins to migrate towards the positive electrode. Then, proteins will agglomerate in a region within the glycine and chloride ions. This region will have low conductance and will allow all the proteins to reach the resolving gel at the same time. Due to the higher pH that the resolving gel possess, the glycine ions will speed up and pass the proteins. Finally, the proteins will start to separate in the resolving gel as a result of the higher conductance.

In order to visualize the proteins after the process of SDS-PAGE, the gel can be stained with Coomassie blue. Coomassie blue will bind to proteins non-specifically and it will be diluted in a methanol and acetic acids mixture. Next, the gel can be transferred to a PVDF membrane, which will help to avoid the binding of non-specific proteins. Finally, an antibody probe will be used to recognize the certain portion of the protein.