Protein analyses with PAGE

All proteins are composed of combinations of the 20 amino acids bound together to form a polypeptide. The amount and combinations of amino acids is what makes proteins different from each other. Proteins therefore differ in terms of their mass and their overall charge. To study proteins, we can compare these properties using a technique known as electrophoresis. Protein analyses by polyacrylamide gel electrophoresis is also known as PAGE.

Protein electrophoresis is a laboratory technique whereby charged (negative or positive) protein molecules are drawn through a solid gel support by an electrical field. The overall charge of the proteins is determined by the unique combination of amino acids.

Native PAGE (NP) is a suitable method for studying the composition and structure of native proteins. During NP proteins are subjected to electrophoresis under conditions where both their conformation and biological activity will remain intact during analyses (non-denaturing technique). Native PAGE can therefore be used to study protein charge, conformation, and mass. The charge of a native protein is pH dependent, and therefore basic knowledge of the protein of interest is needed for choosing the correct buffering system, which ultimately allows for migration of proteins during electrophoresis. The sizes of proteins can be difficult to determine with NP, however, this can give an indication of protein damage/change in conformation.

Sodium dodecyl sulfate (SDS) PAGE allows for separation of proteins based on their size alone. Treatment of samples allows for the proteins to unfold (denature) into a linear shape, whilst evenly coating them with a negative charge. In this way proteins obtain a similar mass to charge ratio, allowing them to migrate toward the positive end (anode) of the gel and be separated based on size. The smaller proteins will migrate faster and further on the gel due to less resistance during electrophoresis.

At Innovative Research Solutions, we have the knowledge and tools to analyse proteins via multiple protein electrophoretic techniques, all in which can give a lot of information about the protein makeup of a sample.

Author: Ms.?Anishka Eksteen?(MSc, Biochemistry – Stellenbosch University)