Polyacrylamide gel electrophoresis, or PAGE, is a laboratory technique used to isolate specific molecules like DNA, RNA, or proteins from a mixture. Its main purpose is to achieve a high degree of purity, separating the target molecule from unwanted contaminants. This method is valuable when the purity of the sample is more important than the total amount recovered. The process allows scientists to proceed with sensitive downstream experiments knowing their starting material is clean.
The Principle of Separation
PAGE separates molecules based on their size using a gel made from polyacrylamide, which acts as a molecular sieve. This gel is formed by polymerizing acrylamide with a cross-linking agent, creating a matrix with a defined pore size. When an electrical current is applied, negatively charged DNA, RNA, or protein molecules move through the gel toward the positive electrode.
The gel’s meshwork impedes the movement of larger molecules more than smaller ones. As a result, smaller molecules travel farther through the gel in a given amount of time, while larger molecules lag behind. This differential movement sorts the molecules by size, creating distinct bands, each containing molecules of a similar length.
The Purification Process
Running the Gel
The process begins by loading the mixed sample into wells at one end of the polyacrylamide gel. This gel is submerged in a buffer solution that conducts electricity. Once loaded, a power supply is connected, establishing an electric field across the gel that drives the migration of the charged molecules.
Visualizing the Target
After electrophoresis is complete, the separated molecules are invisible. To see them, scientists apply a specific stain. For DNA and RNA, fluorescent dyes like SYBR Green or ethidium bromide are used, which bind to the nucleic acids and glow under ultraviolet (UV) light. For proteins, a stain such as Coomassie Brilliant Blue is used, which makes them visible as distinct blue bands.
Excising the Band
With the bands visualized, the next step is to physically isolate the molecule of interest. Using the glowing or colored bands as a guide, a researcher uses a clean scalpel to cut the specific band corresponding to the target molecule’s size out of the gel. This gel slice contains the desired molecules, now separated from contaminants.
Eluting the Molecule
The next step is to extract the purified molecules from the excised gel slice in a process known as elution. A common passive technique is the “crush and soak” method, where the gel slice is broken up and soaked in a buffer, allowing the molecules to diffuse out of the gel matrix. A more active method is electroelution, where an electric current drives the molecules out of the gel and into a collection chamber.
Sample Recovery
Following elution, the purified molecules are in a dilute buffer solution. The final step is to concentrate the sample and remove residual salts from the elution buffer. This is achieved through precipitation using ethanol or isopropanol, after which the sample can be collected by centrifugation, washed, and redissolved in a clean buffer.
Key Applications and Uses
PAGE purification is the method of choice when sample purity is required for a subsequent application. A common use is purifying synthetic DNA oligonucleotides, which are short, custom-made strands of DNA. The chemical synthesis process results in a mixture containing the desired full-length product alongside shorter, incomplete sequences, and PAGE separates the full-length oligo from these contaminants.
Another application is isolating a specific DNA fragment generated through a Polymerase Chain Reaction (PCR). PCR can produce non-specific products in addition to the intended target. By running the PCR mixture on a gel, the correct band can be excised, ensuring that only the desired DNA sequence is used in procedures like DNA cloning or sequencing.
In proteomics, the large-scale study of proteins, PAGE is used to isolate a single protein from a complex cellular extract. A purified protein sample is necessary for analysis by techniques such as mass spectrometry, which identifies proteins based on their mass. Without prior purification by PAGE, the resulting data would be a jumble of signals from many proteins, making identification impossible.
Factors Influencing Purity and Yield
Several factors influence PAGE purification, creating a trade-off between the sample’s purity and the total amount (yield) recovered. The concentration of the polyacrylamide in the gel is a primary variable. Gels with a higher concentration have smaller pores and are better for separating small molecules, while lower concentration gels have larger pores suited for larger molecules. Choosing the correct gel concentration is important for achieving clear separation.
The method used to visualize the bands also has an impact. Stains like ethidium bromide, while effective, can damage the nucleic acid structure when exposed to UV light, which may affect downstream applications. An alternative, UV shadowing, is less damaging but also less sensitive, and the choice of stain depends on the experiment’s sensitivity to sample damage.
PAGE purification yields products of high purity, often exceeding 95%. However, this purity comes at a cost. The multi-step process of excising the band, eluting the molecule, and recovering the sample leads to some loss of material at each stage. Consequently, the final yield is lower compared to other purification methods, a compromise made when the experimental outcome depends on the quality of the starting material.