Polyacrylamide gels are an important tool in biological and chemical research. They are widely used for separating biological molecules such as proteins and nucleic acids. Their ability to sort these molecules has made them valuable for understanding cellular processes and genetic information. Polyacrylamide gel electrophoresis, often called PAGE, is a technique that uses these gels for highly resolved separations of macromolecules.
What are Polyacrylamide Gels?
Polyacrylamide gels are formed through the polymerization of acrylamide and a crosslinking agent, N,N’-methylenebisacrylamide (bis-acrylamide). This process involves a free radical reaction, initiated by ammonium persulfate (APS) and catalyzed by tetramethylethylenediamine (TEMED). The reaction creates a three-dimensional mesh-like network, giving the gel its characteristic transparent, jelly-like consistency.
The physical properties of these gels are directly influenced by the concentration of the monomers. The total monomer concentration (%T) and the percentage of crosslinker (%C) determine the gel’s pore size. A higher %T (greater polymer-to-water ratio) results in smaller average pore sizes, which affects how molecules move through the gel. For instance, a 10-20% gel composition is common for separating proteins.
How They Separate Molecules
Polyacrylamide gels separate molecules based on their electrophoretic mobility, their movement through an electric field. When an electric current is applied across the gel, negatively charged molecules migrate towards the positive electrode, while positively charged molecules move towards the negative electrode. The gel matrix acts like a sieve, impeding the movement of molecules based on their size, shape, and charge.
Smaller molecules navigate through the gel’s pores more easily and travel faster and further. Larger molecules experience more resistance and move more slowly through the matrix. This sieving effect allows for the separation of molecules that differ in size, even by small amounts. Molecular weight, charge, and the applied electric current determine the migration rate and their separation within the gel.
Common Applications in Science
Polyacrylamide gels are used widely in various scientific fields for their high-resolution separation of biological macromolecules. One widespread application is in protein analysis, where they identify proteins, determine their molecular weights, and assess their purity. Researchers can verify if a protein sample contains only the desired protein or if other proteins are present as contaminants.
Historically, polyacrylamide gels were important in DNA sequencing, which determines the order of nucleotides in a DNA molecule. While newer technologies have emerged, the principle of separating DNA fragments by size on polyacrylamide gels was key to early sequencing efforts. In forensic science, these gels are employed for DNA fingerprinting, where unique patterns of DNA fragments from a sample can be compared to known samples, assisting in identification in criminal investigations or paternity tests.
Types of Polyacrylamide Gel Electrophoresis
Various forms of polyacrylamide gel electrophoresis (PAGE) exist, each designed for specific analytical needs. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a widely used technique for separating proteins primarily by their molecular weight. In SDS-PAGE, proteins are denatured (their complex three-dimensional structures are unfolded) and coated with a negatively charged detergent, sodium dodecyl sulfate (SDS). This coating gives all proteins a uniform negative charge-to-mass ratio, ensuring their migration through the gel is almost solely dependent on their size.
Native PAGE, in contrast, separates proteins in their native, folded state, without denaturing agents like SDS. This method is useful for studying the functional properties of proteins or protein complexes, as their original structure and enzymatic activity can be preserved. Another advanced technique is Two-Dimensional PAGE (2D-PAGE), which separates proteins based on two different properties. Proteins are first separated by their isoelectric point (the pH at which a protein has no net charge) in the first dimension, and then by their molecular weight in the second dimension using SDS-PAGE. This allows for the separation of highly complex mixtures, providing a more detailed profile of proteins in a sample.