Gel Filtration (GF)
Since the introduction of Sephadex more than 50 years ago, GF has played a key role in the purification of proteins and enzymes, polysaccharides, nucleic acids, and other biological macromolecules.
GF, also known as size-exclusion chromatography (SEC), is the simplest and mildest of all chromatography techniques and separates molecules based on differences in size.
GF is a non-binding method, so buffer composition does not directly affect separation. This means that separation can be performed in the presence of buffer additives, and within a broad pH and ionic strength. GF gives you the flexibility to choose chromatography conditions that are optimal for your sample and requirements for further purification.
GF can be used for high resolution fractionation or group separation. When choosing a GF medium, consider the aim of the experiment (high resolution fractionation or group separation) and the molecular weights of the target proteins and contaminants to be separated.
GE Healthcare offers a range of Gel Filtration Media and Desalting Products in several formats. Prepacked columns and 96-well filter plates are available that can be used manually, together with a chromatography system, or in high-throughput applications.
High resolution fractionation is used to separate the sample into different components based on size differences (e.g., to separate monomers from aggregates or to perform a molecular weight distribution analysis). The fractionation range of the medium defines the range of molecular weights that should be separable. If high resolution is required, choose a medium with high selectivity (i.e., a steep selectivity curve).
Group separation is used to separate the sample into two major groups; for example, in desalting, to remove salts and other low molecular weight contaminants). The exclusion limit of the media indicates the sizes of the molecules that are excluded from the pores of the matrix and therefore elute in the void volume. A GF medium is chosen so that the larger molecules are eluted in the void volume of the column, and the smaller molecules are retained in the total volume.
Desalting at laboratory scale is a well-proven and simple method to rapidly remove low molecular weight contaminants such as salts or unincorporated labels, and at the same time, transfer the sample into the desired buffer in a single step.