Analysis of biological complexes and proteins involves a complex mixture of varied processes amongst which separation technology stands firm for high quality yield of purified proteins. Gel filtration chromatography is mainly used for separating protein complexes based on molecular size. In this process, the proteins are resolved and separated as they migrate through a chromatographic column which is packed with a gel. This gel consists of a heterogeneous phase of continuous aqueous media which is encompassed within a continuous solid phase, the gel matrix.
The Superdex 200 10/300 GL column from GE Healthcare is a high performance gel filtration column. These are pre-packed glass columns intended for use in gel filtration of proteins, peptides and other biomolecules. Typically, gel filtration finds its application in fractionation by size, MW estimation, separation of monomers and dimers from higher order oligomeric forms, determination of equilibrium constants, determination of molecular weight distribution of polymers, desalting, etc. Other Superdex grades, e.g. Superdex 75, are also available from the same supplier, but have a lower resolution capacity.
The matrix of a chromatographic gel filtration column can be made up of Sephacryl HR, Superdex, Superose, Sephadex, Sepharose or Sepharose CL. Selection of the gel media depends on the intended application of the column. In case of the 10/300 GL column from GE Healthcare, the gel matrix is composed of Superdex, which is based on highly cross-linked agarose beads covalently bonded to dextran molecules. The gel filtration properties of the superdex matrix are governed mainly by the dextran chains.
The Superdex 200 10/300 GL column has a mean bead size 13 um and the fractionation range for the globular proteins is 10,000 – 600,000. The upper exclusion limit for globular proteins that this column can handle is approximately 1.3x106. The bed dimensions for this column are 10 x 300-310 mm and the column bed volume is approximately 24 ml. The column matrix is stable over a pH range of 3-12 and the recommended operating temperature ranges from +4 to +40oC. The storage temperature varies between +4 and +30°C. This column is suitable for in-line use with AKTA or with any other FPLC system (We used Bio-Rad's Biologics DuoFlow System.). The maximum column pressure for the Superdex 200 column is 218 psi, and the recommended flow rate is 0.25-0.75 ml/min, with the maximum flow rate being 1 ml/min. Equilibration of the column must be done with 2 bed volumes of the equilibration buffer before any experiment is performed.
I have used the Superdex 200 10/30 GL gel filtration column from GE Healthcare extensively for my research. My protein complexes were big in size and consisted of a mixture of higher-order oligomeric forms, dimers and monomers. Initially, I standardized the column elution fractions with a standard molecular weight marker kit from Sigma. This gave me a rough idea of where I could expect my protein complexes to elute. Before injecting the sample, I have generally cleaned it and concentrated to a volume of ~ 250 ul. The sample was then injected as a part of the program that was run on the FPLC. Before injection of the sample, I have always equilibrated the column with the recommended volume of equilibration buffer. Elution was generally carried out with a neutral buffer of choice, with the addition of protease inhibitors so as to maintain the integrity of the protein. Post-elution, I have always preferred cleaning the column with at least 2 bed volumes of PBS / water. Long term storage of the column can be done either in 20% degassed ethanol or in any neutral buffer containing 0.02 – 0.05 % sodium azide, which prevents any kind of bacterial growth. Use of any kind of oxidizing agents or unfiltered solutions might be harmful for the column and may lead to clogging of the pores. All solutions used should also be degassed before running through the column.
The Superdex 200 10/300 GL column works efficiently and I have not faced any major problems as such. The only downside is that the column is a bit expensive and in order to use it, the research group should have access to an AKTA or other FPLC system, but these instruments are commonly available for any high-end protein research.