Size exclusion chromatography (SEC) has shown to be a versatile tool for use in a broad range of applications. As buffer conditions do not affect separation, SEC is especially suited for applications including membrane proteins that are difficult to study due to their hydrophobic, poorly soluble nature.
Separating molecules solely based on their size makes SEC well-suited for studies of protein size, monomer stability, and integrity. As SEC can be run under non-denaturing conditions, this technique has also been found to be useful in studies of protein complex formation.
Analysis of membrane proteins using size exclusion chromatography
Unlike ion exchange or affinity chromatography, molecules do not bind to the SEC medium. Instead, SEC (or gel filtration) separates proteins based on their size. As the buffer composition has no direct effect on the separation, conditions can be varied to suit the sample. The possibility of performing analyses under native conditions makes SEC an excellent technique for discrimination between monomer, oligomer, and aggregated forms of a target protein.
Determination of molecular weight
One benefit of using SEC in protein characterization is that the technology can separate a wide range of molecular weights with good resolution (1). In addition, SEC can handle a broad range of polarities, from hydrophilic soluble proteins to hydrophobic membrane proteins. In contrast to SDS-PAGE, SEC analysis includes few manual steps, is simple to use, gives highly reproducible results, can easily be automated, and provides molecular weight data with high precision.
The application note Purification and chromatographic characterisation of an integral membrane protein describes the purification and characterization of recombinant histidine-tagged cytochrome bo3 from E. coli (2). This integral membrane protein was purified as an intact protein complex, including its four subunits as determined by SDS-PAGE analysis. Using SEC, the molecular weight of the entire protein complex could be determined.
Characterization of molecular interactions
As SEC can be used under non-denaturing conditions, elution profiles can be generated to monitor protein interactions and complex formations. The work of Palombo, Daley, and Rapp (3), summarized in the application note Analysis of conformational changes of a membrane protein using size exclusion chromatography (4), shows the usability of SEC in the analysis of the regulation of Mg2+ uptake through the CorA magnesium channel in response to Mg2+ binding. By altering buffer conditions, Mg2+-dependent conformational changes could be studied with 20 mutant proteins in one single 24 h experiment. The method enabled separation of mutants that had lost their ability to sustain the native pentameric state from mutants that with retained ability to form pentamers. By fitting normal distributions to the elution profile, the pentamer content could be calculated for each mutant as the fraction of the total area (monomer + pentamer).
Determination of protein stability
Protein stability is of utmost importance for protein crystallization. SEC is frequently used in investigations of protein assembly and stability. In general, a stable, folded, monomeric protein is shown as one distinct peak in the chromatogram, while an unstable, aggregated, or unfolded protein will give rise to multiple, often asymmetric, peaks. Kawate and Gouaux used fluorescence-detection SEC in precrystallization screenings of membrane proteins derived from prokaryotic or eukaryotic organisms and recombinantly expressed as GFP-fusion proteins (5). The sensitive method allowed the use of nanogram quantities of unpurified protein, and provided data on expression level, degree of monodispersity, and approximate molecular weight of the proteins.
The application note Rapid protein analysis and screening of membrane protein buffer conditions by gel filtration gives a detailed description on how SEC can be used in screening of buffer conditions for obtaining monomer stability of membrane proteins (6).
Improved SEC tools
Superdex 200 Increase and Superose 6 Increase are the new generation agarose-based SEC media, offering higher resolution in shorter period of time compared with the predecessors Superdex 200 and Superose 6, respectively. Superdex 200 Increase is suitable for separation of proteins in the Mr 10 000 to 600 000 molecular weight range, with optimal separation in the molecular weight range for antibodies. Superose 6 Increase is best suited for large proteins and protein complexes, even though the fractionation range is broad, Mr 5 000 to 5 000 000. Superdex 200 Increase and Superose 6 Increase are available prepacked in three chromatography column sizes, each having its own purpose. The 10/300 GL columns are designed for high-resolution SEC at microgram to milligram scale (sample volume 25–500 µl), while the smaller 5/150 GL columns are designed for rapid screening studies (sample volume 4–50 µl). The 3.2/300 columns give high resolution and good sensitivity for small sample volumes (4–50 µl).
Learn more about size exclusion chromatography.
1. Goetz, H., Kuschel, M., Wulff, T., Sauber, C., Miller, C., Fisher, S., and Woodward C. Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination. J Biochem Biophys Methods 60, 281–293 (2004).
2. Application note: Purification and chromatographic characterisation of an integral membrane protein. GE Healthcare, 18112892, Edition AB (2007).
3. Palombo, I., Daley, D.O., Rapp, M. The periplasmic loop provides stability to the open state of the CorA magnesium channel. JBC 287, 27547–27555 (2012).
4. Application note: Analysis of conformational changes of a membrane protein using size exclusion chromatography. GE Healthcare, 29179006, Edition AA (2015).
5. Kawate, T and Gouaux, E. Fluorescence-Detection Size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).
6. Application note: Rapid protein analysis and screening of membrane protein buffer conditions by gel filtration. GE Healthcare, 28932456, Edition AA (2007).