SDS-PAGE – Gel Based Protein Separation

SDS-PAGE is a method of separating proteins based on their molecular mass.  SDS (sodium dodecyl sulfate) is a detergent that binds proteins and covers them with a negative charge.  In general, one SDS molecule binds to two amino acids.  After exposure to SDS different proteins will have very similar charge to mass ratios because  SDS coats the protein in a negative charge overwhelming whatever intrinsic charge the protein originally had. The acronym PAGE stands for polyacrylamide gel electrophoresis.  The protein mixture is denatured by adding SDS and beta-mercaptoethanol (used to break disulfide bonds) and then heated. The denatured protein mixture is added to a polyacrylamide gel where an electric field is applied and the proteins, which are now coated with a negative charge, move towards the positive electrode (the anode).

Proteins move through the gel based on their size.  The polyacrylamide gel can be thought of as a series of nets stacked on top of the each other.  The higher the percentage of polyacrylamide the more nets there are and the size of the holes between the polyacrylamide chains becomes smaller making it more difficult for proteins to move through.  If you want to separate proteins that are relatively small, a higher percentage of polyacrylamide should be used.  If one needs to separate larger proteins a smaller percentage of polyacrylamide would be used. Because the sample buffer contains blue dye which binds to proteins, the proteins that are separated by SDS-PAGE form blue bands which are easily visualized.

SDS-PAGE is often used as a quality control measure for biopharmaceuticals.  A pure protein product with a single chain should show only one band using SDS-PAGE.  Any additional bands would be contaminants or degradation products.  SDS-PAGE is also the first step in creating a western blot which can be used to identify a particular protein within a mixture.

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