Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis(SDS-PAGE) is a commonly applied electrophoretic1 method, which can be used to separate proteins with molecular masses between 5 and 250 kDa (kilodaltons)2. SDS-PAGE allows us to separate proteins based on their molecular masses.
Proteins are polymers of amino acids. Each amino acid carries a charge based on the pH3 of its surrounding medium. Consequently, proteins have distinct charges based on their amino-acid composition. Interaction between the different amino acids of a protein and their charges leads to the protein getting folded.
Adding Sodium Dodecyl Sulphate (SDS), an anionic detergent4, leads to the protein structure getting unfolded. The hydrophobic (water-repelling) chain of SDS interacts with the hydrophobic core of proteins and forces the protein to lose its structure. This is called denaturation.
SDS molecules have negatively charged ends which get attached to the positive charges on proteins. This gives rise to different proteins acquiring a similar charge-to-mass ratio. Reagents such as β-mercaptoethanol may be added to break disulphide bonds in the protein, which cannot be done by SDS.
The SDS-treated protein samples are then placed on a stacking gel made of acrylamide. Gels are a meshwork of polymers with pores in them through which proteins can move. The stacking gel is layered on top of another acrylamide based gel called the resolving gel which has slightly different concentration (and hence pore size) and pH.
The proteins, stacking gel and resolving gel are connected to electrodes and an electric field is applied. The stacking gel ensures that all samples reach the resolving gel at the same time, to account for differences in the time the sample was loaded.
Different proteins carrying different charges traverse dissimilar distances (towards the positive anode, since the samples have negative charge) when subjected to the field. Larger proteins travel less, as they face more resistance in moving through the pores of the resolving gel.
Proteins of different molecular weights appear as distinct bands on the gel, after staining with an appropriate dye such as Coomassie Brilliant Blue (CBB). Proteins with known molecular weights may also be added to the mixture (of proteins) to get a reference point for determining the mass of unknown proteins. This is called a protein ladder as it appears as a series of bands.
SDS-PAGE is a fundamental process in protein purification, that is, separating a desired protein from an impure mixture. Proteins of interest can be located and separated using Western Blotting and Immunocytochemistry.
Proteins with known molecular weights may also be added to the mixture (of proteins) to get a reference point for determining the mass of unknown proteins. This is called a protein ladder
process used to separate DNA, RNA, proteins by applying uniform electric field based on their mass, charge, etc ↩
Dalton(Da) is a unit for measuring small masses, 1 Da = 1 u ↩
pH gives a measure of how acidic/alkaline something is ↩
detergents with anionic part constituting the larger portion of the molecule ↩