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Electrophoresis is used often in protein analysis as well as in nucleic acid analysis. In this technique, small samples of charged molecules are propelled through a matrix by an electric field. Scientists can use electrophoresis to learn more about the composition of a given sample, because different molecules will travel through the matrix at different rates depending on their physical characteristics. Factors that affect the migration rate of proteins during electrophoresis include:

  • Electric field strength
  • Electrophoretic media properties
  • Temperature
  • Buffer concentration
  • Protein charge
  • Protein size and shape

Proteins feature a broad variety of shapes and sizes, and the dissociation constants of their constituent amino acids determine their charges. These properties give proteins characteristic migration rates that researchers and bio-manufacturers exploit for efficient separation. Protein electrophoresis occurs in gel-based or liquid media.

Applications of protein electrophoresis

Common applications of protein electrophoresis are:

  • Sample separation prior to immunoblot analysis
  • Sample analysis prior to downstream applications such as chromatography
  • Determining isoelectric point (pI)
  • Investigating enzymatic activity

Protein electrophoresis methods

Polyacrylamide gel electrophoresis (PAGE)

This technique uses a porous acrylamide gel matrix to separate proteins based on size. Proteins move through a gel in response to electric fields. Smaller molecules move faster than larger ones.


In SDS-PAGE, SDS, a detergent, is added to the acrylamide gel. SDS binds to and denatures proteins, breaking them down into smaller, negatively charged subunits. Because all subunits bound to SDS have a similar negative charge, SDS-PAGE is well suited for separating proteins by size, but not by native net charge.

Native PAGE

Native PAGE doesn’t denature proteins and instead separates them by net charge and shape in addition to size.

Isoelectric focusing (IEF)

IEF separates proteins based on their native isoelectric point (pI). During IEF, proteins migrate through a pH gradient, stopping at their isoelectric points, or the point in the gradient where their net charges are zero.

2-D electrophoresis

2-D electrophoresis separates proteins by size (SDS-PAGE) and by isoelectric point (IEF).

Protein electrophoresis FAQs

Below are answers to some frequently asked questions about protein electrophoresis.

What is protein gel electrophoresis?

Protein gel electrophoresis is a method that helps separate proteins based on mass, shape, and/or charge.

What gel is used in protein electrophoresis?

Polyacrylamide is commonly used for this application. Agarose gels can also be used for larger molecules.

How do you separate proteins by electrophoresis?

There are many protocols and methods for separating proteins by electrophoresis. Download our handbook to learn more about 2-D electrophoresis.