Proteins can be separated based on their isoelectric point and molecular weight using electrophoresis. This allows us to observe and compare structural and functional differences in proteins, and enables further analysis of specific proteins we may want to study.

Separation by Isoelectric Point

Separation by isoelectric point is called isoelectric focusing. In this technique, samples are applied to a well and then run through a pH gradient to which an electrical current is applied. Depending on their charge, proteins will be pulled toward the anode (+) or cathode (-). As the proteins migrate, they eventually settle at the position along the gradient that matches their isoelectric point (0).

Isoelectric focusing

Separation by Molecular Weight

A common method used to separate proteins by molecular weight is SDS-PAGE. SDS-PAGE stands for sodium dodecyl sulphate–polyacrylamide gel electrophoresis. SDS is a detergent that denatures protein converting their tertiary structure to peptide chains with a high negative charge. The peptides are then separated by running them through a polyacrylamide gel with the application of an electrical current. The negatively charged peptides move from the negative to the positive electrode, with the smallest fragments migrating the fastest.

Two-Dimensional Gel Electrophoresis

A technique that takes advantage of the above two methods, is two-dimensional gel electrophoresis. One disadvantage that you can imagine with isoelectric focusing is that more than one protein can have the same isoelectric point. Therefore, they will occupy the same position on the pH gradient after separation. Therefore, to allow further separation, the partially separated proteins can be added to an SDS-PAGE apparatus where they are separated based on size. On the other hand, you can also figure that even if you start with SDS-PAGE, the resulting protein bands may be composites of proteins that have the same molecular weight. Hence, separation of those bands by another dimension, i.e., pI, would be necessary.

Courtney Simons
Courtney Simons is a food science professor. He holds a BS degree in food science and a Ph.D. in cereal science from North Dakota State University.
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