SDS-PAGE is a very commonly used technique used in the lab for the separation of proteins based on their molecular weight. SDS-PAGE separates the proteins based on their rates of migration through a gel under the influence of electricity.

 

The movement of anything charged through an electric field is determined by its net charge. However, the problem with naturally folded proteins is that they don’t have a charge and their size is dependent on their weight. So a protein’s charge is determined by adding up all the positive and negative charges of the amino acidsA simple organic compound that make up proteins in the protein and the size is determined by the proteins three-dimensional structure. So proteins in their naturally folded state would move at different speeds based off of their charge and shape. However, SDS-PAGE aims to separate proteins based off their weight only. In order to separate the proteins by weight only, the three-dimensional structure must be broken.

 

The SDS part of SDS-PAGE is a chemical that when boiled along with the 3-D protein can unfold the protein from a 3-D structure to a linear structure. SDS also coats this linear protein with negative charges. The SDS is also present in the gel, to make sure that the proteins don’t lose their negative charge and stay linear.

 

Once the protein has been unfolded and made negative, it is loaded into a gel which in this case is a polyacrylamide gel (PAGE). The gel isn’t reactive and it can be made at different porosities. In the case for these experiments, the gels were 12% and 15%. The higher porosity allows the bigger proteins to travel further down the gel.

 

The loaded gels are placed into a specialized container with a buffer. As the electricity runs, the negatively charged proteins in the wells of the gel move downwards, towards the positively charged end. The more porous the gel, the more the larger proteins can move further down the gel.