Acrylamide Gel Electrophoresis: How the Process Works

Acrylamide gel electrophoresis is a laboratory method used to separate molecules like proteins and nucleic acids based on their size and electrical charge. This technique is a widespread practice for analyzing complex mixtures in biochemical research.

The Science Behind Separation

The technique forces molecules through a polyacrylamide gel, which acts as a molecular sieve. When an electric field is applied, charged molecules move through the gel’s pores. The direction of movement is dictated by the molecule’s charge, with negative molecules moving toward the positive electrode and vice versa.

Smaller molecules navigate the pores more easily and travel farther through the gel in a given amount of time. Larger molecules are impeded by the mesh-like structure and move more slowly. This difference in mobility allows for effective separation based on size.

For protein analysis, the detergent sodium dodecyl sulfate (SDS) is used. SDS denatures proteins into linear chains and coats them with a uniform negative charge. This process masks their natural charges, so the proteins separate on the gel almost exclusively based on their molecular weight.

Essential Components and Their Functions

The gel is formed by polymerizing acrylamide with a cross-linking agent, bis-acrylamide. The concentration of acrylamide determines the gel’s pore size. Higher concentrations create smaller pores for small molecules, while lower concentrations yield larger pores for bigger molecules.

Polymerization is initiated by ammonium persulfate (APS) and catalyzed by tetramethylethylenediamine (TEMED). Buffer solutions maintain a stable pH, which affects molecular charge, and conduct the electrical current. A discontinuous buffer system, using different buffers in the gel and tank, produces sharper bands.

Physical equipment includes a gel casting apparatus of glass plates and spacers to mold the gel. A comb is inserted into the gel solution before it solidifies to create wells for loading samples. The solidified gel is placed in an electrophoresis tank with buffer and electrodes connected to a power supply that generates the electric field.

Visualizing the Invisible: The Electrophoresis Workflow

The process begins by pouring the prepared polyacrylamide gel solution between two glass plates. Two layers are poured: a lower “separating” gel for the main separation, and an upper “stacking” gel. The stacking gel has larger pores to concentrate the sample into a narrow band before it enters the separating gel.

While the gel polymerizes, samples are prepared by mixing them with a loading buffer. This buffer contains SDS to denature proteins, glycerol to help the sample sink into the wells, and a tracking dye to monitor progress. The prepared samples are then loaded into the gel’s wells.

With samples loaded, the chamber is filled with running buffer and connected to a power supply. An electric current causes the molecules to migrate through the gel toward the positive electrode. Since the separated molecules are invisible, the gel is soaked in a stain, like Coomassie Brilliant Blue for proteins, which reveals them as distinct bands.

What We Learn from the Bands

The result is a pattern of bands on the gel, each representing molecules of the same size. A primary application is determining molecular weight by running a “molecular weight marker,” a mix of known-sized molecules, in an adjacent lane. By comparing the distance an unknown molecule traveled to the markers, its size can be estimated.

Sample purity can also be assessed. A purified protein sample should appear as a single band. The presence of multiple bands indicates the sample contains several different proteins, visually representing its complexity.

The technique allows for comparing different samples side-by-side on the same gel. A researcher might compare protein extracts from healthy and diseased tissue to identify differences in protein presence or quantity. This comparison can provide clues about the molecular basis of a disease or confirm the success of genetic engineering.

Safety First: Handling Acrylamide

Acrylamide requires careful handling due to its toxicity. In its unpolymerized powder or liquid form, acrylamide is a neurotoxin and a potential human carcinogen. Direct contact and inhalation of the powder must be avoided.

To mitigate risks, laboratory personnel must use personal protective equipment (PPE), including nitrile gloves, a lab coat, and safety glasses. All work with unpolymerized acrylamide should be performed inside a chemical fume hood to prevent inhaling dust or aerosols.

Proper disposal is also a safety component. Liquid waste with unpolymerized acrylamide and contaminated materials must be disposed of as hazardous chemical waste. Once polymerized into a gel, acrylamide is largely non-toxic because the monomers are locked in the polymer matrix. It is still good practice to handle finished gels with gloves, as trace amounts of unpolymerized material may remain.