Determination of Molecular Size by Size‐Exclusion Chromatography (Gel Filtration)

G. Brent Irvine1

1 Queen's University Belfast, Medical Biology Centre, Belfast
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 5.5
DOI:  10.1002/0471143030.cb0505s06
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Size‐exclusion or gel filtration chromatography is one of the most popular methods for determining the sizes of proteins. Proteins in solution, or other macromolecules, are applied to a column with a defined support medium. The behavior of the protein depends on its size and that of the pores in the medium. If the protein is small relative to the pore size, it will partition into the medium and emerge from the column after larger proteins. Besides a protein's size, this technique can also be used for protein purification, analysis of purity, and study of interactions between proteins. In this unit protocols are provided for size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and for conventional gel filtration, including calibration of columns (in terms of the Stokes radius) using protein standards.

PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: SE‐HPLC
  • Basic Protocol 2: Conventional SEC
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: SE‐HPLC

  • Mobile phase (see recipe)
  • Total volume (V t) marker (see recipe)
  • Void volume (V o) marker (see recipe)
  • Protein calibration standards (see recipe)
  • Solution in mobile phase of protein whose size is to be determined
  • 0.22‐µm filters (protein‐compatible type for solutions containing protein)
  • HPLC system, comprising injector, one pump, UV‐detector, and chart recorder or integrator
  • Prepacked SE‐HPLC column (Table 5.5.1) with appropriate fractionation range (see Strategic Planning)
  • Guard column
  • 10‐ to 100‐µl syringe compatible with injector used
  • Filtration apparatus
  • Vacuum pump
  • Fraction collector

Basic Protocol 2: Conventional SEC

  • Mobile phase for conventional SEC (see recipe)
  • Gel filtration support (Table 5.5.2) with appropriate fractionation range (see Strategic Planning)
  • Colored marker (see recipe)
  • Total volume (V t) marker (see recipe)
  • Protein calibration standards (see recipe)
  • Solution in mobile phase of protein whose size is to be determined
  • 0.22‐µm filters (protein‐compatible type for solutions containing protein)
  • Buchner funnel and flask
  • Vacuum pump
  • Empty column (see Strategic Planning) with column fittings and packing reservoir
  • 10‐ml syringe
  • Peristaltic pump
  • UV‐detector
  • Chart recorder or integrator
  • Filtration apparatus
  • Fraction collector
  • Sample applicator
PDF or HTML at Wiley Online Library


  •   FigureFigure 5.5.1 Separation of a mixture of seven solutes on a Zorbax Bio‐series GF‐250 column. 20 µl of a mixture containing ∼1.5 µg of each protein was injected. The solutes were, in order of elution, thyroglobulin, alcohol dehydrogenase, ovalbumin, myoglobin, insulin, glucagon and sodium azide. The number beside each peak is the elution time in minutes. The absorbance of the highest peak, insulin, was 0.105. The equipment was a model 501 pump, a 441 absorbance detector operating at 214 nm, a 746 data module, all from Waters and a Rheodyne model 7125 injector with 20‐µl loop. The column was a Zorbax Bio‐Series GF‐250 with guard column (Dupont). The mobile phase was 0.2 M sodium phosphate buffer, pH 7.0, and the flow rate was 1 ml/min. This figure is reproduced from Irvine with permission from Humana Press, Totowa, N.J.
  •   FigureFigure 5.5.2 Plots of calibration curves for standard proteins. The data were obtained on the column and under the conditions described in Figure . The protein standards were thyroglobulin, apoferritin, yeast alcohol dehydrogenase, serum albumin, ovalbumin, myoglobin, and insulin. Stokes radii for these proteins are listed in Table (except for alcohol dehydrogenase, with RS = 4.55 nm). (A) A plot of RS versus Kd in which all the data were used. (B) A plot of log RS versus Kd. In the latter plot the top and bottom values of Kd (thyroglobulin and insulin) were omitted. Vo was determined to be 6.67 ml from the elution peak of blue dextran. Vt was determined to be 12.28 ml from the elution peak of sodium azide.


Literature Cited

Literature Cited
   Ackers, G.K. 1970. Analytical gel chromatography of proteins. Adv. Protein Chem. 24:343‐446.
   Dubin, P.L. and Principi, J.M. 1989. Failure of universal calibration for size exclusion chromatography of rodlike macromolecules versus random coils and globular proteins. Macromolecules 22:1891‐1896.
   Fish, W.W., Reynolds, J.A., and Tanford, C. 1970. Gel chromatography of proteins in denaturing solvents. J. Biol. Chem. 245:5166‐5168.
   Hagel, L. 1989. Gel filtration. In Protein Purification: Principles,High Resolution Methods and Applications (J.C. Janson and L. Ryden, eds.) pp. 63‐106. VCH Publishers, New York.
   Hagel, L. 1992. Peak capacity of columns for size‐exclusion chromatography. J. Chromatogr. 591:47‐54.
   Himmel, M.E. and Squire, P.G. 1981. High pressure gel permeation chromatography of native proteins on TSK‐SW columns. Int. J. Pept.Protein Res. 17:365‐373.
   Horiike, K., Tojo, H., Yamano, T., and Nozaki, M. 1983. Interpretation of Stokes radius of macromolecules determined by gel filtration chromatography. J. Biochem. 93:99‐106.
   Irvine, G.B. 1996. Molecular weight estimation for native proteins using size‐exclusion high‐performance chromatography. In The Protein Protocols Handbook (J.M. Walker, ed.) pp. 473‐478. Humana Press, Totowa, N.J.
   Irvine, G.B. and Shaw, C. 1986. High performance gel permeation chromatography of proteins and peptides on columns of TSK‐G2000‐SW and TSK‐G3000‐SW. Anal. Biochem. 155:141‐148.
   Le Maire, M., Aggerbeck, L.P., Monteilhet, C., Andersen, J.P., and Moller, J.V. 1986. The use of high‐performance liquid chromatography for the determination of size and molecular weight of proteins: A caution and a list of membrane proteins suitable as standards. Anal.Biochem. 154:525‐535.
   Le Maire, M., Ghazi, A., Moller, J.V., and Aggerbeck, L.P. 1987. The use of gel chromatography for the determination of sizes and relative molecular masses of proteins. Biochem. J. 243:399‐404.
   Le Maire, M., Ghazi, A., Martin, M., and Brochard, F. 1989. Calibration curves for size‐exclusion chromatography: Description of HPLC gels in terms of porous fractals. J.Biochem. 106:814‐817.
   Moore, J.C., 1964. Gel permeation chromatography. I. A new method for molecular weight distribution of high polymers. J. Polymer Sci. A2:835‐843.
   Nave, R., Weber, K., and Potschka, M. 1993. Universal calibration of size‐exclusion chromatography for proteins in guanidinium hydrochloride including the high‐molecular‐mass proteins titin and nebulin. J. Chromatogr. 654:229‐246.
   Nozaki, Y., Schechter, N.M., Reynolds, J.A., and Tanford, C. 1976. Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemistry 15:3884‐3890.
   Pfannkoch, E., Lu, K.C., Regnier, F.E., and Barth, H.G. 1980. Characterization of some commercial high performance size‐exclusion chromatography columns for water‐soluble polymers. J. Chromatogr. Sci. 18:430‐441.
   Pharmacia Biotech 1996. Gel Filtration Principles and Methods, 6th ed. Lund, Sweden.
   Porath, J. and Flodin, P. 1959. Gel filtration: A method for desalting and group separation. Nature 183:1657‐1659.
   Potschka, M. 1987. Universal calibration of gel permeation chromatography and determination of molecular shape in solution. Anal. Biochem. 162:47‐64.
   Regnier, F.E. 1983. High‐performance liquid chromatography of proteins. Methods Enzymol. 91:137‐190.
   Shaw, C. 1997. Purification of extracted peptides for structural analysis. In Neuropeptide Protocols; Methods in Molecular Biology, Vol. 73 (G.B. Irvine and C.H. Williams, eds.) pp. 9‐16. Humana Press Totowa, N.J.
   Tanford, C. 1961. Physical Chemistry of Macromolecules. John Wiley & Sons, New York.
   Tanford, C., Nozaki, Y., Reynolds, J.A., and Makino, S. 1974. Molecular characterization of proteins in detergent solutions. Biochemistry 13:2369‐2374.
   Wen, J., Arakawa, T., and Philo, J.S. 1996. Size‐exclusion chromatography with on‐line light‐scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal.Biochem. 240:155‐166.
   Wu, C.‐S. 1999. Column Handbook for Size Exclusion Chromatography. Academic Press, San Diego.
Key References
   Ackers, 1970. See above.
  A detailed and rigorous treatment of theory.
   Hagel, 1989. See above.
  A comprehensive review with emphasis on protein SEC.
   Pharmacia Biotech 1996. See above.
  Of most use for the manufacturer's own products, but has much good general practical advice on packing columns and is illustrated with many diagrams and photographs.
PDF or HTML at Wiley Online Library