Introduction to Protein Electrophoresis

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.

Related Topics: Protein Transfer, Immunodetection, and Imaging.

 

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How Protein Electrophoresis Works

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).

Diagram of protein migration in electrophoresis

Movement of proteins during electrophoresis.

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.

 
General Considerations in Protein Electrophoresis

A electrophoresis workflow involves the selection of the appropriate method, instrumentation, and reagents for the intended experimental goal.

Methods include the following:

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.

 
Protein Electrophoresis Workflow

Diagram of protein electrophoresis workflowMethod SelectionSample PreparationReagent Selection and PreparationPerforming ElectrophoresisProtein Detection and Analysis

A typical protein electrophoresis workflow.

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References for Protein Electrophoresis

Dunn MJ (1993). Gel electrophoresis of proteins (Abingdon Oxford, United Kingdom: BIOS Scientific Publishers Ltd.).

Garfin DE (1990). One-dimensional gel electrophoresis. Methods Enzymol 182, 425–441.

Documents MADHA

Number Description Options
6040
Electrophoresis Guide, Interactive PDF, Rev C
2651
2-D Electrophoresis Workflow How-To Guide, Rev F
2895
Protein Blotting Guide, Ver C