SDS PAGE Protocol -MChem Project v1

SDS-PAGE is a widely-used technique to analyse proteins. The system consists of two gels - a Resolving Gel (aka Running Gel) that separates proteins based on Molecular Weight (Mw), and a Stacking Gel. Proteins are aliquoted into wells and concentrated before resolution. The differences in the composition between the gels and between the electrophoresis buffer produce a system capable of finely resolving proteins based on Mw. This protocol describes the process of making a 15% Acrylamide Gel, a commonly used %age gel. The %age of Polyacrylamide in the gel governs the size of the pores, so the higher the %age of Acrylamide the smaller the sizes of the pores and therefore the smaller the species are that can be observed. Background: The tertiary structure of proteins means that proteins are often folded with a variety of geometries and due to the hydrophobic effect, their surfaces are usually coated with charged amino acids. Because of this, the molecular weight of folded proteins would be impossible to determine from their relative migration in an electric field. To solve this problem, proteins are resolved after converting proteins into a uniform unfolded state by the addition of SDS. SDS is an anionic surfactant, which binds to all the positive and coats the protein in a negative charge. SDS application causes the proteins to lose their tertiary structure and become linear. Mercaptoethanol is used in the loading buffer, which contributes to the linearisation of proteins by breaking disulphide bonds found in the tertiary structure. After becoming coated in a negative charge, the proteins will have a similar net negative charge and therefore a similar mass: charge ratio. As a result of this, the proteins would resolve depending on their mass, not their charge. Glycine is key to the discontinuous buffer system of SDS-PAGE. It is a Zwitterion, at low pH more Glycine molecules become cationic, while at high pH more molecules become anionic. When an electric field is applied, Glycinate anions move towards the stacking layer of a lower pH and become more neutrally charged. They move more slowly through the stacking layer toward the anode. In contrast, anions such as Chloride from Tris-HCl move more quickly toward the anode. Chloride and Glycine zwitterions hit the loading wells and create a steep and narrow voltage gradient between the Chloride ion front (Leading front) and the neutral Glycine zwitterion front (Trailing front). Protein samples are therefore concentrated into this zone and ushered through the stacking layer. In the resolving front, with the higher pH, the Glycine ions become negatively charged and with the Chloride ions, move toward the anion quickly. The ions are unaffected by the Acrylamide and deposit the proteins in a tight band on the resolving layer, with the smaller proteins having moved down the furthest/quickest.