Western Blotting Electrophoresis Techniques

Electrophoresis Basics

How Protein Electrophoresis Works

The term electrophoresis refers to the movement of charged molecules in response to an electric field. Electrophoresis has been developed into a standard molecular biology laboratory technique, where molecules of different sizes and charges are transported through a medium at different rates by an applied electrical field, resulting in their separation.

In an electric field, proteins move toward the electrode of opposite charge. Proteins have a wide range of sizes and shapes and have charges imparted to them by their constituent amino acids. As a result, proteins have characteristic migration rates that can be exploited for separation. The total charge on a protein in an aqueous medium depends on the degree of ionization of its acid and base groups, which can be controlled by buffering. Because the mass of a protein is constant, its charge-to-mass ratio is thereby altered. Protein conformation can also be changed by buffering, detergents, and oxidation/reduction. These factors allow for differences in protein migration rates to be tuned to a degree, thus enhancing separation. In protein electrophoresis, the proteins move through and aqueous polymer gel, which retards migration and retains them for imaging and analysis. The rate proteins move in gel electrophoresis is governed by relationships among the characteristics of the electrophoresis system and the proteins.

Factors affecting protein migration include:

• Gel composition and strength (weight percentage of polymer)
• Electric field strength
• Temperature
• Protein size, shape, and charge — which are in turn affected by
  • Buffer pH, ion type, and concentration
  • Addition of detergents or reducing agents

Polyacrylamide Gel Electrophoresis (PAGE) Overview

Almost all methods for protein separation use an aqueous polyacrylamide gel as a size-selective sieve during separation. This polyacrylamide gel is what lends the name to the technique: polyacrylamide gel electrophoresis, or PAGE. As proteins move through the gel in response to an electric field, the gel’s pore structure allows smaller proteins to travel more rapidly than larger proteins. This difference in migration rate between smaller and larger proteins leads to their physical separation in the gel.

In most PAGE applications, the gel is mounted between two buffer chambers, and the only electrical path between the two buffers is through the gel. Usually, the gel has a vertical orientation, and the gel is cast with a comb that creates wells in which the samples are deposited. Applying an electrical field across the buffer chambers forces the migration of proteins into and through the gel. The movement of the proteins through the gel is normally tracked visually by the addition of a dye to the sample, which can be seen moving further down the gel over time. If the gel is run for too long, the proteins can run off the gel.

Schematic of electrophoretic protein separation in a polyacrylamide gel. MW, molecular weight.

Native PAGE

Native PAGE is a method in which proteins are prepared in nonreducing, nondenaturing sample buffer, and electrophoresis is also performed in the absence of denaturing and reducing agents. Since the native charge-to-mass ratio is preserved, protein mobility is determined by a complex combination of factors. Additionally, since protein-protein interactions are retained during separation, some proteins may also migrate together as multisubunit complexes and move in unpredictable ways. Moreover, because the native charge is preserved, proteins can migrate towards either electrode depending on their charge.

Effect of SDS on proteins in aqueous buffer. SDS helps denature proteins so they adopt a rod-like conformation. SDS also adds an overall negative charge to the protein.

Migration of Proteins and Ions

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  Denatured proteins are loaded into the wells.

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  Voltage applied and proteins move into gel. Chloride ions present in gel (leading ions) run faster than SDS-bound proteins and form an ion front. Glycinate ions (trailing ions) flow in from running buffer and form a front behind proteins.

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  Proteins are stacked between chloride and glycinate ion fronts. Percentage of acrylamide increases and the pore size decreases at interface between stacking and resolving gels. Movement of proteins into the resolving gel is met with increased resistance.

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  Proteins are stacked between chloride and glycinate ion fronts. Percentage of acrylamide increases and the pore size decreases at interface between stacking and resolving gels. Movement of proteins into the resolving gel is met with increased resistance.

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  The smaller pore size of the resolving gel separates the proteins. Since the charge-to-mass ratio is equal in all the proteins of the sample, separation is based on molecular weight only.

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  Individual proteins are separated into bands according to their molecular weights.

SDS-PAGE

This method incorporates the detergent sodium dodecyl sulfate (SDS) into the buffer system and has become the most popular form of protein electrophoresis. When proteins are separated in the presence of SDS and denaturing agents, for example, reducing agents to dissociate intra-protein disulfide bonds, they become fully denatured and dissociate from each other. In addition, SDS binds noncovalently to proteins and imparts several characteristics that can be exploited for protein separation:

• Since SDS is negatively charged, it masks the intrinsic charge of the protein and provides an overall negative charge to the proteins
• A similar charge-to-mass ratio for all proteins in a mixture, since SDS binds at a consistent rate of 1.4 gram of SDS per gram of protein
• A long, rod-Iike shape of the proteins instead of a complex tertiary conformation

As a result, the rate at which SDS-bound protein migrates in a gel depends primarily on its size, enabling molecular weight estimation of the individual proteins as they are resolved into separate bands on the gel.

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