Gel electrophoresis can be used to separate proteins for both analysis and purification. This section provides a brief overview of the theory and workflow of protein electrophoresis.
The rate at which proteins move in an electrical field (migration rate, in units of cm2 V-1 sec-1) is governed by a complex relationship between the physical characteristics of both the electrophoresis system and the proteins. The strength of the electric field, the properties of the electrophoretic medium (usually a polyacrylamide gel), the temperature of the system, and the pH, ion type, and concentration of the buffer all play roles along with the size, shape, and charge of the proteins (Garfin 1990). Proteins come in a wide range of size and shapes and have charges determined by the dissociation constants of their constituent amino acids. As a result, proteins have characteristic migration rates that can be exploited for separation purposes. Protein electrophoresis can be performed in liquid or gel-based media and can also be used to move proteins from one medium to another (for example, in blotting applications).
Protein electrophoresis can be used in a variety of applications including protein purification or purity determination (for example, at various stages during a chromatographic purification), to determine size, isoelectric point (pI), and enzymatic activity, or to provide data on the regulation of protein expression (Dunn 1993). In fact, a number of different techniques can be grouped under the term protein electrophoresis including gel electrophoresis, isoelectric focusing (IEF), and two-dimensional (2-D) electrophoresis.
A electrophoresis workflow involves the selection of the appropriate method, instrumentation, and reagents for the intended experimental goal.
Methods include the following:
- Various types of polyacrylamide gel electrophoresis (PAGE)
- Instrumentation needed for protein electrophoresis includes:
Once proteins have been separated, they can be utilized for a number of downstream applications including enzymatic assays, further purification, transfer to a membrane for immunological detection (immunoblotting or western blotting), and elution and digestion for mass spectrometric analysis.