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Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography

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Protein Science
Aug 2016



Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of Rs by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure.

This protocol aims to describe a gel filtration experiment on a prepacked column using a Fast Protein Liquid Chromatography (FPLC) system to determine the Rs of proteins with some indications that are specific for Ca2+ sensor proteins.

Keywords: Size exclusion chromatography (分子排阻色谱法), Gel filtration (凝胶过滤), Hydrodynamics (流体动力学), Stokes radius (斯托克斯半径), Protein shape (蛋白质形状), Protein size (蛋白质大小), Conformational change (构象改变), Ca2+-sensor proteins,


Gel filtration separates molecules of different sizes and shapes based on their relative abilities to penetrate a bed of porous beads with well-defined pore sizes, which identifies the fractionation range. Molecules larger than the fractionation range, which are completely excluded from entering the pores, flow quickly through the column and elute first at the void volume (V0), which is the interstitial volume outside the support particles. Molecules smaller than the fractionation range, which are able to diffuse into the pores of the beads, have access to the total volume available to the mobile phase, therefore they move through the bed more slowly and elute last. Molecules with intermediate dimensions will be eluted with an elution volume (Ve) comprised between the void volume and the total volume available to the mobile phase (the smaller the molecule, the greater its access to the pores of the matrix, and thus the greater is its Ve).

The molecular weight of a protein can be determined by comparison of its elution volume parameter Kd, which represents the distribution of a given solute between the stationary and mobile phases (see Data analysis below), with those of different known calibration standards.

If the protein of interest has the same shape (generally globular) as the standard calibration proteins, the gel filtration experiment provides a good estimate of its molecular weight. However, because the shape of proteins can vary significantly and may be not known for an unknown protein, care must be taken in the determination of molecular size from elution volume. For example, a protein with an elongated shape could elute at a position that does not correspond to its dimension and which is significantly different from the position of a spherical protein having the same molecular weight. This is the case for some Ca2+ sensor proteins, e.g., calmodulins from different organisms (Sorensen and Shea, 1996; Sorensen et al., 2001; Astegno et al., 2014; Astegno et al., 2016; Vallone et al., 2016), which have anomalous migrations in gel filtration, resulting in a defined overestimation of the molecular weight due to their highly extended conformation. Thus, it is clear that in a gel filtration experiment the elution profile of proteins is closer to their Stokes radius (Rs) rather than to their molecular weight. Rs is a hydrodynamic value indicative of the apparent size of the dynamic solvated/hydrated protein.

For this reason, a SEC-based approach has been employed to resolve Ca2+-induced changes in the hydrated shape of Ca2+ sensor proteins by determination of their Rs in apo- and Ca2+-bound conditions. Ca2+ binding usually causes a decrease in the Rs (Sorensen and Shea, 1996; Sorensen et al., 2001; Astegno et al., 2016). The same SEC-based approach may be applicable to the detection of other protein-small molecule (e.g., other metals) interactions that cause changes in the structure of the protein to a less or more extended conformation (Asante-Appiah and Skalka, 1997; Bagai et al., 2007; De Angelis et al., 2010).

The values of Rs have been reported for a large number of proteins; in particular, some proteins are especially convenient for calibration of gel filtration columns (le Maire et al., 1986; Uversky, 1993) (Table 1). A gel filtration column can determine the hydrodynamic size, Rs, of a sample protein by comparison with the Rs of these water-soluble calibration proteins.

Table 1. Standards for calibrating analytical gel filtration

Materials and Reagents

  1. 0.22 µm syringe filters with low protein retention (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: 42204-PV )
  2. 0.22 µm vacuum filtration unit (Sartorius, catalog number: 180C7-E )
  3. Superdex 200 10/300 GL prepacked column (GE Healthcare, catalog number: 17517501 )
  4. Superose 12 10/300 GL prepacked column (GE Healthcare, catalog number: 17-517-301 )
  5. 20% EtOH–minimum 1 L (Sigma-Aldrich, catalog number: 51976 )
  6. MilliQ water–minimum 1 L
  7. Protein markers:
    Thyroglobulin (Sigma-Aldrich, catalog number: T9145 )
    Apo-Ferritin (Sigma-Aldrich, catalog number: A3660 )
    β-amylase (Sigma-Aldrich, catalog number: A8781 )
    Catalase (Sigma-Aldrich, catalog number: C9322 )
    Aldolase (Sigma-Aldrich, catalog number: A2714 )
    Alcohol dehydrogenase (Sigma-Aldrich, catalog number: A8656 )
    Albumin (Sigma-Aldrich, catalog number: A8531 )
    Ovalbumin (Sigma-Aldrich, catalog number: A5503 )
    Carbonic anhydrase (Sigma-Aldrich, catalog number: C7025 )
    Myoglobin (Sigma-Aldrich, catalog number: M0630 )
    Cytochrome c (Sigma-Aldrich, catalog number: C7150 )
  8. Gel filtration Calibration Kits with an optimized range of proteins are also commercially available:
    1. Gel Filtration Markers Kit for Protein Molecular Weights 29,000-700,000 Da (Sigma-Aldrich, catalog number: MWGF1000 )
    2. Gel Filtration Markers Kit for Protein Molecular Weights 12,000-200,000 Da (Sigma-Aldrich, catalog number: MWGF200 )
    3. Gel Filtration Markers Kit for Protein Molecular Weights 6,500-66,000 Da (Sigma-Aldrich, catalog number: MWGF70 )
    4. Gel Filtration Calibration Kits
      High molecular weight (GE Healthcare, catalog number: 28-4038-42 )
      Low molecular weight (GE Healthcare, catalog number: 28-4038-41 )
  9. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: 21097 )
  10. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E4378 )
  11. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 1.06462 )
  12. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
  13. DL-dithiothreitol, anhydrous (DTT) (Sigma-Aldrich, catalog number: D9779 )
  14. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 31248 or P9333 )
    Note: The product “ 31248 ” has been discontinued.
  16. HEPES (Sigma-Aldrich, catalog number: H3375 )
  17. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  18. Acetone (Sigma-Aldrich, catalog number: 439126 )
  19. Dextran blue (Sigma-Aldrich, catalog number: D4772 )
  20. 2 M CaCl2 (20 ml) (see Recipes)
  21. 0.1 M EGTA (100 ml, pH 8) (see Recipes)
  22. 0.1 M DTT (see Recipes)
  23. Mobile phase (see Recipes)
    1. 5 mM Tris, 150 mM KCl, pH 7.5 (for calibration of Superose 12 column 10/300GL)
    2. 5 mM Tris, 150 mM KCl, 5 mM EGTA, 1 mM DTT pH 7.5
    3. 5 mM Tris, 150 mM KCl, 5 mM CaCl2, 1 mM DTT pH 7.5
    4. 50 mM HEPES, 150 mM NaCl, 0.1 mM DTT pH 7.5 (for calibration of Superdex 200 10/300 GL)
  24. Total volume available to the mobile phase marker (see Recipes)
  25. Void volume marker (see Recipes)
  26. Protein calibration standards (see Recipes)
  27. Protein sample (see Recipes)


  1. ÄKTA FPLC system (or similar liquid chromatography system, e.g., ÄKTA pure , ÄKTA prime plus , ÄKTA start ) including injector, one pump, UV-detector, Fraction collector (GE Healthcare, models: ÄKTA pure , ÄKTA prime plus , ÄKTA start )
    Note: ÄKTA FPLC has been discontinued and replaced with ÄKTA pure .
  2. Sample loop kit (GE Healthcare, catalog number: 18-0404-01 )
  3. Hamilton syringe (500 µl) (Hamilton, catalog number: 81217 or Sigma-Aldrich, catalog number: S9266 )
  4. Vacuum pump
  5. Tabletop centrifuge (Eppendorf, model: 5424 R )


  1. UNICORNTM 4.0 control software for ÄKTA FPLC chromatography system (GE Healthcare)
  2. Origin 8.0 (OriginLab Corporation, Northampton, MA)


The following protocol describes the determination of Rs by using a prepacked column in a typical FPLC system (Figure 1). Buffers and other solutions are delivered via a system pump, and the sample can be load in different ways (e.g., by a syringe and a sample loop or by a sample pump). A detection system (e.g., UV/Vis absorbance, conductivity) is located after the column to control the separation process and the proteins are collected in the fraction collector. It is possible to operate the system manually, although the system is usually controlled by software. In particular, in the ÄKTA FPLC all data and parameters of the separation run are displayed in the System Control module of UNICORN software, which permits control of chromatography systems (e.g., system settings, run data, curves) and on-line monitoring of separation processes. Refer to the UNICORN User Reference Manual for software functionality.

Figure 1. Typical FPLC system. A. Scheme of basic components and typical flow path for a chromatography system; B. Picture of GE Healthcare ÄKTA FPLC apparatus.

  1. Filter and de-gas all solutions (MilliQ water, eluent, 20% ethanol) through a 0.22 µm filter.
  2. Move the input lines of FPLC system from 20% EtOH and place them into MilliQ water.
    Note: Make sure that the back-pressure over the column does not exceed the maximum pressure value mandated by the manufacturer.
  3. Connect the column (injector outlet–column inlet and column outlet–detector) (Figure 2).
    Note: Make sure that there is no air in the valves and tubing prior to connecting the column to a chromatography system. Ensure that the inlet of the column is filled with liquid; connect it to the system in a drop-to-drop fashion (unscrew the inlet of the column, let the buffer flow through the system and drop to the inlet of the column; then, unscrew the outlet of the column and connect to the detector).

    Figure 2. Chromatography column connection. A. Detailed view of Superose 12 10/300 GL prepacked column connected to ÄKTA FPLC system and of some important components of the instrument, i.e., mixer valve, injection valve, sample loop, and detectors. B. Detail of the column outlet and its connection to the detectors.

  4. When using the column after prolonged storage or for the first time equilibrate the column as follows:
    1. At least 2 times the volume of the column (2x column volumes) of MilliQ water at a flow rate of 0.5 ml/min. Columns are usually stored in 20% ethanol, which produce high back pressures, so the initial flow rates should not exceed half the maximum recommended flow rate. Since ethanol might cause precipitation of the salts used in the mobile phase, it is advisable to flow MilliQ water until complete ethanol removal before proceeding with the equilibration.
    2. 2x column volumes of mobile phase (eluent) at a flow rate of 0.5-1 ml/min.
      Note: The eluent should be chosen to ensure full sample solubility. In order to avoid any unwanted ionic interactions between the matrix and the solute molecules, the use of a buffer with an ionic strength equivalent or greater to 0.15 M NaCl or KCl is recommended. The manufacturer usually provides a list of some useful eluent compositions for a given column.
  5. Connect the loop (100 μl loop size) to injection valve. Wash the loop by injection with MilliQ water (at least 5 times the volume of the loop). Repeat the wash with the protein’s eluent (according to the column used).
  6. Prepare the samples and remove particulate material from protein solution by either centrifuging (10,000 x g for 15 min) or filtering samples through a 0.22 µm filter.
  7. The sample is injected through the injection valve which has two positions, load and inject. When the valve is set on load, the loop can be filled with the sample (Figure 3A, blue line) and the eluent is being bypassed to the column (Figure 3A, green line). When the valve is set on inject, the eluent flows through the loop and moves the sample to the column (Figure 3B, green line).

    Figure 3. Schematic representation of the injection valve in the load (A) and inject (B) positions

  8. Determine the void volume by loading blue dextran (200-300 μl at 1-2 mg/ml dissolved in the mobile phase, see Recipes) to the column via loop. This concentration of blue dextran will give an absorbance at 280 nm (A280) of 0.5-1 in the peak fraction. Dextran blue can also be used to control column packing.
    Note: An excess of sample is used to ensure that the sample loop is filled completely (complete filling method). To achieve 95% of maximum loop volume about 2 to 3 loop volumes of sample are necessary. Before injection, make sure the flow rate is correctly set and that the injection valve is set to ‘load’. Load the syringe with the sample (2-3 times the loop volume) and expel the air bubbles. It may be necessary to flick the syringe to push the bubbles up. Gently inject the syringe contents into the loop. Leave the syringe in position and set the injection valve to ‘Inject’. The ideal volume loads correspond to sample volumes between 1-2% of the total column volume to obtain sharp peaks. Additionally, solutions containing high protein concentrations (> 2 mg/ml) can lead to increased viscosity and hence decreased diffusion coefficients of solutes (Ricker and Sandoval, 1996).The typical flow rate is 0.5-1 ml/min, although by decreasing the flow rate in SEC experiments, greater resolution can be obtained on a given column (refer to the manufacturer’s recommendation for flow rate).
  9. Inject an aliquot of 200-300 μl of acetone at 5 mg/ml (0.63% v/v dissolved in the mobile phase, see Recipes) into the column to determine the total volume available to the mobile phase. Detection of acetone is possible at 280 nm.
  10. Prepare a mixture of selected standard proteins by dissolving them in the eluent buffer at 0.5-1 mg/ml (or at concentrations indicated by the manufacturer, see Recipes). Check for protein complete dissolution before applying to the column and then determine Ve for each protein standard.
    Note: Typically, five protein standards are sufficient to obtain a good calibration plot. It is possible to use more than one standard in the same run (only if the peaks of the different standards are completely resolved). The standards must be chosen accurately, taking into account the fractionation range of the selected column.
  11. Apply the protein sample (200-300 μl at 1 mg/ml) with the unknown Rs and determine the corresponding Ve.
    Note: For determination of Ca2+-dependent change in Rs of Ca2+ sensor proteins, the sample protein must be loaded first in the absence and then in the presence of Ca2+. Apo conditions include 5 mM Tris, 150 mM KCl, 1 mM DTT, and 5 mM EGTA at pH 7.5, while Ca2+-saturated conditions include 5 mM Tris, 150 mM KCl, 1 mM DTT, and 5 mM CaCl2 at pH 7.5.
  12. If the column has to be stored, wash it with 2x column volumes of MilliQ water and then at least 2x column volumes of 20% ethanol. Store at 4 °C.
    Note: For column data, cleaning-in-place conditions, column performance controls, buffers, and solvent resistance always refer to the manufacturer’s instructions.

Data analysis

  1. Results in gel filtration are typically visualized as a plot of the volume of the mobile phase eluted through the column versus the detector signal (for proteins usually signal at 280 nm) which is called chromatogram. The elution volume of a given molecule is determined by calculating the volume of eluent from the point of injection to the center of the elution peak.
    Note: Peak integration in the chromatography software is used to identify and measure curve characteristics, such as elution volumes, peak areas, and peak widths. In particular, to perform a basic peak integration in the Evaluation module of Unicorn software 4.0:
    Select the Integrate:Peak Integrate menu commandSelect a source curveSelect Calculate baseline in the Baseline list. The peaks in the integrated chromatogram are automatically labelled with their elution volumes. Several other peak characteristics are also calculated automatically. To display other characteristics, select the options that you want to display from the Select peak table columns list.
  2. The distribution coefficient Kd, which represents the fraction of the stationary phase available for diffusion of a given solute species, can be expressed as follows:

    where (see also Figure 4),
    Ve is the elution volume for a particular protein,
    V0 is the void volume for the column, i.e., the elution volume of molecules totally excluded from the pores of the beads,
    Vi is the volume of the solvent inside the matrix and is equal to the volume of the stationary phase.
    In the method described in this protocol, Vi is determined by the measurement of the elution volume of acetone (i.e., the maximum retention volume experienced by a small molecule that has access to the total volume available to the mobile phase, since it will distribute freely between the mobile and stationary solvent phases) minus the void volume V0.
    However, for the molecular weight and/or Rs estimations in SEC, it is widely accepted to substitute the term Vt - Vo for Vi and use the partition coefficient (Kav):

    Vt is the total volume of the packed bed column (Figure 4) and is measured by geometrically calculating the volume of a cylinder, (πr2h, where r and h are the radius and the length of the column, respectively, both expressed in cm).
    Vt represents the sum of the total volume available to the mobile phase and the solid support volume, which is not accessible to solvent. Since the estimated volume of the stationary phase (Vt - Vo) will include the volume of the gel-forming substance, Kav is not a true distribution coefficient. However, Kav is directly proportional to Kd and, like Kd, defines solute behavior independently of the bed dimensions and packing (Figure 4) (Irvine, 2001 and GE Healthcare Handbook).

    Figure 4. Gel filtration column parameters. A. Schematic representation of V0, Vt, and Vi, which are depicted as grey areas. B. Theoretical chromatogram for a gel filtration experiment with the summary of the various gel filtration parameters.

  3. Calculate Kd for the standards and obtain a calibration curve by plotting Kd versus the logarithm of Rs (for Rs values see Table 1). An example of column calibration (Superdex 200 HR 10/300 GL prepacked column) obtained using a commercially available gel filtration calibration kit is shown in Figure 5. The calibration curve is prepared by plotting the Kd value for each standard versus its corresponding log10 Rs value. An essentially linear relationship should be obtained. Once the Kd value is calculated for the unknown sample, its Rs value can be determined from the calibration graph. Data were analyzed using Origin 8.0 (OriginLab Corporation, Northampton, MA).
    Note: Plotting elution volumes vs. Rs for the standard proteins is usually adequate, and usually produces an approximately linear plot.

    Figure 5. Superdex 200 HR 10/300 GL prepacked column (GE Healthcare) for size exclusion chromatography. Chromatographic separation and calibration curve for standard proteins. For this gel filtration analysis the mobile phase is 50 mM HEPES, 150 mM NaCl, 0.1 mM DTT pH 7.5. A. Overlap of chromatograms obtained from two runs (solid and dotted lines) of standard proteins. 1. Thyroglobulin; 2. Ferritin; 3 β-amylase; 4. Alcohol dehydrogenase; 5. Albumin bovine serum; 6. Carbonic anhydrase; 7. Cytochrome c. B. Calibration curve prepared by plotting the Kd value for each standard in (A) versus its corresponding log10 Rs value used to determine Rs of the protein sample. Calibration data are from (Astegno et al., 2015).

Practical example: determination of Ca2+-dependent changes in the Rs of the Ca2+-sensor protein CaM1 from Arabidopsis thaliana
SEC was used to determine Ca2+-induced variations in the hydrated shape of CaM1 from Arabidopsis thaliana (AtCaM1) (Astegno et al., 2016). A Superose 12 10/300 GL prepacked column was calibrated with standard proteins using 5 mM Tris, 150 mM KCl, pH 7.5 as the mobile phase (Figure 6A). The hydrodynamic behavior of AtCaM1 was then analyzed in the absence and presence of Ca2+ by adding 5 mM EGTA or 5 mM CaCl2, respectively, to the mobile phase and to the sample. Superposition of the chromatograms of AtCaM1 in the presence and the absence of Ca2+ (Figure 6B) indicates that the Ve of AtCaM1 is increased when the protein is in the Ca2+-bound form. The calibration curve was then used to evaluate the Rs of Ca2+-free and Ca2+-bound states of AtCaM1 and shows that the addition of Ca2+ to AtCaM1 is accompanied by a decrease in its Rs value (Figure 6C) as a consequence of the large conformational change that AtCaM1 undergoes upon Ca2+ binding. AtCaM1 is a monomer in both the apo and Ca2+-bound forms at the concentrations used in this study. The Rs values were derived from at least 3 replicates for each protein sample and mean ± SD were calculated.

Figure 6. Determination of Rs for apo- and Ca2+-bound CaM1 from Arabidopsis thaliana. The Rs difference between the Ca2+-free and Ca2+-bound states of AtCaM1 was determined using a Superose 12 column 10/300GL (GE Healthcare). A. Overlap of chromatograms obtained from two runs (solid and dotted lines) of standard proteins. 1. Albumin bovine serum; 2. Ovalbumin; 3. Carbonic anhydrase; 4. Myoglobin; 5. Cytochrome c. B. Elution profiles of AtCaM1 in the absence (apo-AtCaM1) and presence (Ca2+-AtCaM1) of Ca2+; C. Calibration curve prepared by plotting the Kd value for each standard in (A) versus its corresponding log10 Rs value. The graph was used to determine the Rs of AtCAM1 in apo- and Ca2+-bound conditions. The average Rs of apo- and Ca2+-AtCaM1 at pH 7.5 was 2.77 ± 0.03 nm and 2.53 ± 0.04 nm, respectively, and for both states results in molecular weight overestimation. Moreover, Ca2+ binding to AtCaM1 results in a decrease of Rs (~0.24 nm). Experimental data are from (Astegno et al., 2016).


  1. Instability of the column temperature negatively affects reproducibility of separation and column efficiency. We recommend that you keep the column at controlled temperature (usually 25 °C).
  2. Avoid extreme changes in pH or other conditions that can cause protein inactivation or even precipitation. If the sample precipitates in the SEC column, the column will be blocked, possibly irreversibly, and the sample might be lost.
  3. SEC columns must not run dry. Ensure that there is sufficient buffer for long runs. Columns that run dry must be repacked.
  4. Bubble formation on mixing of solvents can lead to many problems in FPLC analysis (e.g., appearance of spurious peaks, decrease in pumping reliability) which can be prevented by thoroughly degassing all solutions.


  1. 2 M CaCl2 (20 ml)
    Dissolve 5.88 g of CaCl2 in 20 ml of MilliQ water
  2. 0.1 M EGTA (100 ml, pH 8)
    Dissolve 3.8 g of EGTA in about 20 ml MilliQ water, bring to pH 11 with NaOH and then to pH 8.0 with HCl. Finally, add MilliQ water to a final volume of 100 ml
  3. 0.1 M DTT
    Dissolve 0.46 g of DTT (DL-dithiothreitol, anhydrous) in 25 ml of deionized or distilled H2O , adjust volume to 30 ml, dispense into aliquots, and store at -20 °C
  4. Mobile phase
  5. Mobile phase marker
    Dissolve acetone in the mobile phase at 5 mg/ml (0.63 % v/v). Centrifuge at 10,000 x g for 15 min immediately before column loading
  6. Void volume marker
    Prepare a solution of blue dextran (average MW 2,000,000 Da) by dissolving it at 1-2 mg/ml in the mobile phase. Centrifuge at 10,000 x g for 15 min immediately before column loading
  7. Protein calibration standards
    Solubilize each protein standard in the mobile phase at a concentration 1-2 mg/ml or as specified in the manufacturer’s instructions without heating or mixing vigorously. Centrifuge at 10,000 x g for 15 min immediately before column loading
  8. Protein sample
    Dissolve the protein sample in the mobile phase at 1 mg/ml (for determination of Ca2+-dependent change in Rs of Ca2+ sensor proteins add 5 mM EGTA or 5 mM CaCl2 to the sample)


This protocol was adapted from the previously published studies by Uversky, 1993; Sorensen and Shea, 1996; Astegno et al., 2016; Vallone et al., 2016.


  1. Asante-Appiah, E. and Skalka, A. M. (1997). A metal-induced conformational change and activation of HIV-1 integrase. J Biol Chem 272(26): 16196-16205.
  2. Astegno, A., Capitani, G. and Dominici, P. (2015). Functional roles of the hexamer organization of plant glutamate decarboxylase. Biochim Biophys Acta 1854(9): 1229-1237.
  3. Astegno, A., La Verde, V., Marino, V., Dell'Orco, D. and Dominici, P. (2016). Biochemical and biophysical characterization of a plant calmodulin: Role of the N- and C-lobes in calcium binding, conformational change, and target interaction. Biochim Biophys Acta 1864(3): 297-307.
  4. Astegno, A., Maresi, E., Marino, V., Dominici, P., Pedroni, M., Piccinelli, F. and Dell'Orco, D. (2014). Structural plasticity of calmodulin on the surface of CaF2 nanoparticles preserves its biological function. Nanoscale 6(24): 15037-15047.
  5. Bagai, I., Liu, W., Rensing, C., Blackburn, N. J. and McEvoy, M. M. (2007). Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system. J Biol Chem 282(49): 35695-35702.
  6. De Angelis, F., Lee, J. K., O'Connell, J. D., 3rd, Miercke, L. J., Verschueren, K. H., Srinivasan, V., Bauvois, C., Govaerts, C., Robbins, R. A., Ruysschaert, J. M., Stroud, R. M. and Vandenbussche, G. (2010). Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems. Proc Natl Acad Sci U S A 107(24): 11038-11043.
  7. Ge Healthcare Life Science Handbook: Size Exclusion Chhromatography. Principles and Methods.
  8. Irvine, G. B. (2001). Determination of molecular size by size-exclusion chromatography (gel filtration). Curr Protoc Cell Biol Chapter 5: Unit 5 5.
  9. le Maire, M., Aggerbeck, L. P., Monteilhet, C., Andersen, J. P. and Møller, 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(2): 525-535.
  10. Ricker, R. D. and Sandoval, L. A. (1996). Fast, reproducible size-exclusion chromatography of biological macromolecules. J Chromatogr A 743(1): 43-50.
  11. Sorensen, B. R., Eppel, J. T. and Shea, M. A. (2001). Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse. Biochemistry 40(4): 896-903.
  12. Sorensen, B. R. and Shea, M. A. (1996). Calcium binding decreases the stokes radius of calmodulin and mutants R74A, R90A, and R90G. Biophys J 71(6): 3407-3420.
  13. Vallone, R., La Verde, V., D'Onofrio, M., Giorgetti, A., Dominici, P. and Astegno, A. (2016). Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana. Protein Sci 25(8): 1461-1471.
  14. Uversky, V. N. (1993). Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule. Biochemistry 32(48):13288-98.


尺寸排阻色谱法(SEC)或凝胶过滤是一种流体动力学技术,它将溶液中的分子作为其尺寸和形状的函数。在蛋白质的情况下,可以通过实验得出的流体动力学值是斯托克斯半径(R s),它是具有相同流体力学性质的球体的半径(即, >摩擦系数)作为生物分子。通过SEC测定R s已经被广泛用于监测由钙(Ca 2+)与许多Ca 2+连接引起的构象变化传感器蛋白。对于这类蛋白质,SEC分离不仅基于Ca 2 + 结合后的蛋白质尺寸变化,而且可能来自水合壳结构的变化。
该方案旨在使用快速蛋白质液相色谱(FPLC)系统对预填充柱进行凝胶过滤实验,以确定蛋白质的R 1,其中一些适用于Ca 2 + 传感器蛋白。

凝胶过滤基于其相对的能力分离不同大小和形状的分子,以穿透具有明确孔径的多孔珠床,其识别分馏范围。大于完全排除进入孔隙的分馏范围的分子快速流过色谱柱,首先以空间体积(V 0 O)(其为支持颗粒外的间隙体积)洗脱。能够扩散到珠的孔中的小于分级范围的分子具有可用于流动相的总体积,因此它们更缓慢地移动通过床并最后洗脱。具有中等维度的分子将以包含在流动相可利用的空隙体积和总体积之间的洗脱体积(V e e e e)被洗脱(分子越小,其进入孔隙越大矩阵,因此其V e e越大)。
蛋白质的分子量可以通过比较其洗脱体积参数K 来确定,其表示在固定相和流动相之间的给定溶质的分布(参见下面的数据分析),其中那些不同的已知校准标准。
如果感兴趣的蛋白质具有与标准校准蛋白质相同的形状(通常为球状),则凝胶过滤实验提供了其分子量的良好估计。然而,由于蛋白质的形状可以显着变化并且对于未知蛋白质可能是未知的,因此必须注意从洗脱体积测定分子大小。例如,具有细长形状的蛋白质可以在与其尺寸不对应的位置洗脱,并且与具有相同分子量的球形蛋白质的位置显着不同。来自不同生物体的钙调蛋白(例如,Sorensen和Shea,1996; Sorensen等人)的情况就是这样的一些传感器蛋白质例如 ,2001; Astegno等人,2014年; Astegno等人,2016; Vallone等人,2016),其具有异常在凝胶过滤中迁移,由于其高度扩展的构象,导致对分子量的定义过高估计。因此,很明显,在凝胶过滤实验中,蛋白质的洗脱曲线更接近于它们的斯托克斯半径(R s),而不是它们的分子量。 R sub是表示动态溶剂化/水合蛋白质的表观尺寸的流体动力学值。
因此,采用基于SEC的方法,通过测定Ca 2+ +传感器蛋白质的水合形状来测定Ca 2+ / apo和Ca 2 + - 条件下的R 。 Ca 2 + 结合通常导致R sub的降低(Sorensen和Shea,1996; Sorensen等人,2001; Astegno >等,,2016)。相同的基于SEC的方法可能适用于检测导致蛋白质结构变化到更少或更多扩展构象的其他蛋白质 - 小分子(例如,其他金属)相互作用( Asante-Appiah和Skalka,1997; Bagai等人,2007; De Angelis等人,2010)。

已经报告了大量蛋白质的Rs值;特别地,一些蛋白质对凝胶过滤柱的校准特别方便(le Maire等人,1986; Uversky,1993)(表1)。凝胶过滤柱可以通过与这些水溶性校准蛋白质的R sub比较来测定样品蛋白质的流体动力学尺寸R sub。


关键字:分子排阻色谱法, 凝胶过滤, 流体动力学, 斯托克斯半径, 蛋白质形状, 蛋白质大小, 构象改变


  1. 0.22μm注射器过滤器,具有低蛋白质保留(Thermo Fischer Scientific,Thermo Scientific TM,目录号:42204-PV)
  2. 0.22μm真空过滤装置(Sartorius,目录号:180C7-E)
  3. Superdex 200 10/300 GL预填充柱(GE Healthcare,目录号:17517501)
  4. 超级12 10/300 GL预填充柱(GE Healthcare,目录号:17-517-301)
  5. 20%EtOH-1L(Sigma-Aldrich,目录号:51976)
  6. MilliQ水最小1 L
  7. 蛋白质标记:
  8. 凝胶过滤校准试剂盒具有优化的蛋白质范围也可商购:
    1. 蛋白质分子量凝胶过滤标记试剂盒29,000-700,000 Da(Sigma-Aldrich,目录号:MWGF1000)
    2. 蛋白质分子量凝胶过滤标记试剂盒12,000-200,000 Da(Sigma-Aldrich,目录号:MWGF200)
    3. 用于蛋白质分子量的凝胶过滤标记试剂盒6,500-66,000Da(Sigma-Aldrich,目录号:MWGF70)
    4. 凝胶过滤校准套件
      高分子量(GE Healthcare,目录号:28-4038-42)
      低分子量(GE Healthcare,目录号:28-4038-41)
  9. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:21097)
  10. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E4378)
  11. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:1.06462)
  12. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758)
  13. 无水二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
  14. Trizma ®基质(Sigma-Aldrich,目录号:T1503)
  15. 氯化钾(KCl)(Sigma-Aldrich,目录号:31248或P9333)
  16. HEPES(Sigma-Aldrich,目录号:H3375)
  17. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  18. 丙酮(Sigma-Aldrich,目录号:439126)
  19. 葡聚糖蓝(Sigma-Aldrich,目录号:D4772)
  20. 2 M CaCl 2(20 ml)(参见食谱)
  21. 0.1M EGTA(100ml,pH8)(参见食谱)
  22. 0.1 M DTT(见配方)
  23. 流动相(见食谱)
    1. 5mM Tris,150mM KCl,pH7.5(用于校准Superose 12色谱柱10/300GL)
    2. 5mM Tris,150mM KCl,5mM EGTA,1mM DTT pH7.5
    3. 5mM Tris,150mM KCl,5mM CaCl 2,1mM DTT pH7.5
    4. 50 mM HEPES,150 mM NaCl,0.1 mM DTT pH 7.5(用于校准Superdex 200 10/300 GL)
  24. 可用于流动相标记的总体积(见配方)
  25. 空隙体积标记(见配方)
  26. 蛋白质校准标准(见配方)
  27. 蛋白质样品(见食谱)


  1. ÄKTAFPLC系统(或类似的液相色谱系统,例如,ÄKTA纯,ÄKTA素加,ÄKTA启动),包括注射器,一个泵,紫外检测器,分馏收集器(GE Healthcare,型号:ÄKTA纯,ÄKTAprime plus,ÄKTA开始)
  2. 样品环套件(GE Healthcare,目录号:18-0404-01)
  3. 汉密尔顿注射器(500μl)(Hamilton,目录号:81217或Sigma-Aldrich,目录号:S9266)
  4. 真空泵
  5. 台式离心机(Eppendorf,型号:5424 R)


  1. UNICORN TM 4.0ÄKTAFPLC色谱系统(GE Healthcare)的控制软件
  2. Origin 8.0(OriginLab Corporation,Northampton,MA)


以下协议通过在典型的FPLC系统中使用预包装的列来描述R> s的确定(图1)。缓冲液和其它溶液通过系统泵递送,并且样品可以以不同的方式(例如,通过注射器和样品环或通过样品泵)进行加载。检测系统(例如,UV/Vis吸光度,电导率)位于柱之后,以控制分离过程,并将蛋白质收集在馏分收集器中。尽管系统通常由软件控制,但是可以手动操作系统。特别是在ÄKTAFPLC中,分离运行的所有数据和参数都显示在UNICORN软件的系统控制模块中,这样可以控制色谱系统(例如,系统设置,运行数据,曲线)和在线监测分离过程。有关软件功能,请参阅UNICORN用户参考手册。

图1.典型的FPLC系统。 A.色谱系统的基本组分方案和典型流路方案; B. GE HealthcareÄKTAFPLC仪器的图片。

  1. 通过0.22μm过滤器过滤和去除所有溶液(MilliQ水,洗脱液,20%乙醇)
  2. 将FPLC系统的输入线从20%EtOH移至MilliQ水中。
  3. 连接色谱柱(注射器出口 - 入口和出口检测器)(图2) 注意:在将色谱柱连接到色谱系统之前,请确保阀门和管道中没有空气。确保色谱柱入口充满液体;将其连接到系统中,以一种即插即用的方式(拧下色谱柱的入口,让缓冲液流过系统并落到色谱柱入口处;然后拧下色谱柱的出口并连接到检测器)。

    图2.色谱柱连接。 A.超级12 10/300 GL预填充柱与ÄKTAFPLC系统和仪器的一些重要组件 连接的详细视图。 ,混合阀,注射阀,样品回路和检测器。 B.色谱柱出口的细节及其与检测器的连接。

  4. 长时间储存​​后使用色谱柱或首次平衡色谱柱如下:
    1. 以0.5ml/min的流速至少2倍体积的MilliQ水(2x柱体积)。色谱柱通常储存在20%乙醇中,产生高背压,因此初始流速不应超过最大推荐流速的一半。由于乙醇可能会导致流动相中使用的盐沉淀,因此建议在进行平衡之前将MilliQ水直至完全乙醇清除。
    2. 2x柱体积的流动相(洗脱液),流速为0.5-1ml/min。
      注意:应选择洗脱液以确保样品溶解度充分。为了避免基质和溶质分子之间的任何不需要的离子相互作用,推荐使用离子强度等于或大于0.15M NaCl或KCl的缓冲液。制造商通常提供给定列的一些有用的洗脱剂组合物的列表。
  5. 将回路(100μl回路尺寸)连接到进样阀。用MilliQ水注射洗涤环(至少是循环体积的5倍)。用蛋白质洗脱液重复洗涤(根据使用的色谱柱)。
  6. 通过离心(10,000×g×15分钟)或通过0.22μm过滤器过滤样品,准备样品并从蛋白质溶液中除去颗粒物质。
  7. 样品通过具有两个位置,注入和注入的注射阀注入。当阀门处于负载状态时,环路可以填充样品(图3A,蓝色线),洗脱液被旁路到柱(图3A,绿线)。当阀门设置在注射器上时,洗脱液流过循环并将样品移动到柱(图3B,绿线)。


  8. 通过加载蓝色葡聚糖(200-300μl,1-2 mg/ml溶于流动相,参见食谱)确定空隙体积。该浓度的蓝色葡聚糖将在峰值分数中在280nm(A <280)处获得0.5-1的吸光度。葡聚糖蓝也可用于控制柱填充。
    注意:使用过量的样品来确保样品环完全填充(完全填充方法)。为达到最大循环体积的95%,需要大约2至3个循环体积的样品。注射前,请确保流量正确设定,并将进样阀设置为"加载"。将样品装入注射器(循环体积的2-3倍)并排出气泡。可能需要轻按注射器才能将气泡向上推。将注射器内容物轻轻注入循环。将注射器放在适当的位置,并将注射阀设置为"注入"。理想的体积载荷对应于总柱体积的1-2%之间的样品体积,以获得尖锐的峰值。另外,含有高蛋白质浓度(> 2mg/ml)的溶液可以导致粘度增加,从而降低溶质的扩散系数(Ricker和Sandoval,1996)。典型的流速为0.5-1ml/min,尽管通过减少在SEC实验中的流速,可以在给定的列上获得更高的分辨率(参见制造商对流速的建议)。
  9. 以5mg/ml(溶解在流动相中的0.63%v/v,见食谱)的200-300μl丙酮注入等分试样,以确定可用于流动相的总体积。丙酮可以在280nm检测
  10. 通过将选定的标准蛋白质溶解在0.5-1mg/ml的洗脱液缓冲液中(或制造商指示的浓度,参见食谱)来制备所选标准蛋白质的混合物。在应用于色谱柱之前检查蛋白质完全溶解,然后确定每个蛋白质标准品的V e。
  11. 应用蛋白质样品(200-300μl,1 mg/ml)和未知的R</sub>,并确定相应的V e 。
    注意:为了测定Ca 2 + 传感器蛋白质的Ca 2 + 依赖性变化,样品蛋白必须首先在不存在的情况下加载,然后在存在Ca 2 + 。 Apo条件包括pH7.5的5mM Tris,150mM KCl,1mM DTT和5mM EGTA,而Ca 2+饱和条件包括5mM Tris,150mM KCl,1mM DTT,和pH7.5的5mM CaCl 2。
  12. 如果色谱柱必须储存,用2x柱体积的MilliQ水洗涤,然后至少2x柱体积的20%乙醇洗涤。储存于4°C。


  1. 凝胶过滤的结果通常可视化为通过色谱柱洗脱的流动相的体积与检测器信号(对于蛋白质通常在280nm的信号)的曲线图,其被称为色谱图。通过计算从注射点到洗脱峰中心的洗脱液体积来确定给定分子的洗脱体积。
    选择Integrate:Peak Integrate菜单命令选择源曲线 在"基准"列表中选择"计算基准"。积分色谱中的峰自动用洗脱体积标记。自动计算其他几个峰值特征。要显示其他特征,请从"选择峰表列"列表中选择要显示的选项。
  2. 表示可用于给定溶质物质扩散的固定相的分数的分布系数K d可以表示如下:

    V 是特定蛋白质的洗脱体积,
    V 是柱的空隙体积,即,完全从珠的孔中排除的分子的洗脱体积,
    在该方案中描述的方法中,通过测量丙酮的洗脱体积来确定V i,即,由具有(1)的小分子经历的最大保留体积获得可用于流动相的总体积,因为它将在移动和固定溶剂相之间自由分配)减去空隙体积V 0 。
    然而,对于SEC中的分子量和/或R sub估计,广泛接受的是将项V 0替代为V 使用分区系数(K av ):

    V 3是填充床柱的总体积(图4),并且通过几何计算圆筒的体积(πr 2 h,其中r和h分别是柱的半径和长度,均以cm表示)。
    V 表示可用于流动相的总体积和固体支持体积的总和,其不能溶于溶剂。由于固定相的估计体积(V sub-V o O)将包括凝胶形成物质的体积,因此K av不是真实的分布系数。然而,K av>>>>> to to>> and and and and>> 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4((( )(Irvine,2001和GE Healthcare Handbook)。

    图4.凝胶过滤柱参数。 A.V <0>,V 和V i 的示意图, ,其被描绘为灰色区域。 B.凝胶过滤实验的理论色谱图,各种凝胶过滤参数的总结。

  3. 计算标准品的K ,并通过绘制K 相对于R s的对数获得校准曲线(对于R</sub>值见表1)。使用市售的凝胶过滤校准试剂盒获得的柱校准(Superdex 200 HR 10/300 GL预填充柱)的实例显示在图5中。校准曲线通过将K 值绘制每个标准对应于其对应的对数的10 值。应该获得基本上线性的关系。一旦为未知样本计算了K 值,则可以从校准图确定其R sub值。使用Origin 8.0(OriginLab Corporation,Northampton,MA)分析数据。

    图5. Superdex 200 HR 10/300 GL预填充柱(GE Healthcare)用于尺寸排阻色谱。标准蛋白的色谱分离和校准曲线。对于该凝胶过滤分析,流动相为50mM HEPES,150mM NaCl,0.1mM DTT pH7.5。 A.从两次运行(固体和虚线)的标准蛋白质获得的色谱图重叠。甲状腺球蛋白铁蛋白3β-淀粉酶;酒精脱氢酶;白蛋白牛血清;碳酸酐酶;细胞色素c。 B.通过绘制(A)中的每个标准与其对应的用于确定的对应的log 10的值的标准曲线准备的校准曲线蛋白质样品中的R 。校准数据来自(Astegno等人,2015)。

实践示例: Ca 2 中的Ca 2 依赖变化的确定+ - 传感器蛋白质CaM1来自拟南芥
使用SEC来确定来自拟南芥(AtCaM1)(AtCaM1)(Astegno等人)的CaM1的水合形状的Ca 2+ 2 +诱导的变化。 2016)。使用5mM Tris,150mM KCl,pH7.5作为流动相,用标准蛋白校准Superose 12 10/300 GL预填充柱(图6A)。然后在不存在和存在Ca 2+的情况下分别向流动相中加入5mM EGTA或5mM CaCl 2,分析AtCaM1的流体动力学行为,例子。存在和不存在Ca 2+的AtCaM1的色谱图叠加(图6B)表明当蛋白质在Ca中时,AtCaM1的V e e升高 2 + - 表格。然后使用校准曲线来评估AtCaM1的Ca 2+和无钙和Ca 2+的平衡状态的R sub,并且表明由于AtCaM1在Ca 2+上经历的大的构象变化的结果,向AtCaM1添加Ca 2 + 伴随着其R sub值的降低(图6C) sup> 2 + 绑定。在本研究中使用的浓度下,AtCaM1是载脂蛋白和Ca 2+/+ +/- 形式的单体。每个蛋白质样品至少有3个重复序列得到R</sub>值,并计算平均值±SD。

图6.来自拟南芥的apo和Ca 2 + - 结合CaM1的R 的测定使用Superose 12色谱柱10/300GL测定AtCaM1的Ca 2 + -free和Ca 2 + - 状态之间的R < (GE Healthcare)。 A.从两次运行(固体和虚线)的标准蛋白质获得的色谱图重叠。白蛋白牛血清;卵清蛋白碳酸酐酶; 4.肌红蛋白细胞色素c。 B.在不存在(apo-AtCaM1)和Ca 2+的存在下(Ca 2 + -AtCaM1)的AtCaM1的洗脱曲线。 C.通过绘制(A)中的每个标准对其相应的对数的10 值的K d d值的准备曲线。该图用于确定apo和Ca 2 + - 条件下AtCAM1的R sub。在pH7.5时apo和Ca 2+的平均值分别为2.77±0.03nm和2.53±0.04nm,两种状态下的分子重量过高估计。此外,与AtCaM1结合的Ca 2 + 导致R sub(〜0.24nm)的降低。实验数据来自(Astegno等人,2016)。


  1. 色谱柱温度的不稳定性对分离和色谱柱效率的再现性产生不利影响。我们建议您将色谱柱保持在受控温度(通常为25°C)。
  2. 避免pH或其他可能导致蛋白质失活甚至沉淀的条件的极端变化。如果样品在SEC色谱柱中沉淀,色谱柱将被阻挡,可能不可逆转,样品可能会丢失。
  3. SEC色谱柱不能干燥。确保长时间有足够的缓冲区。运行干燥的色谱柱必须重新包装。
  4. 混合溶剂时的气泡形成可能会导致FPLC分析中的许多问题(例如,假峰的出现,泵送可靠性的降低),这可以通过彻底脱气所有溶液来防止。


  1. 2 M CaCl 2(20ml)
    将5.88g CaCl 2溶于20ml MilliQ水中
  2. 0.1M EGTA(100ml,pH8)
    将3.8g的EGTA溶解在约20ml的MilliQ水中,用NaOH调至pH11,然后用HCl调至pH8.0。最后,加入MilliQ水至100 ml的最终体积
  3. 0.1 M DTT
    将0.46g DTT(DL-二硫苏糖醇,无水)溶于25ml去离子水或蒸馏水中,调节体积至30ml,分配至等分试样,并储存于-20℃, >
  4. 移动阶段
  5. 流动相标记
    以5mg/ml(0.63%v/v)溶于流动相中的丙酮。在加载柱子之前立即以10,000 x g离心15分钟
  6. 无效体积标记
    通过在流动相中以1-2mg/ml溶解蓝色葡聚糖(平均MW 2,000,000Da)来制备溶液。在加载柱子之前立即以10,000 x g离心15分钟
  7. 蛋白质校准标准
    在流动相中溶解每种蛋白质标准品,浓度为1-2 mg/ml或按照制造商的说明书规定,不要剧烈加热或混合。在加载柱子之前立即以10,000 x g离心15分钟
  8. 蛋白质样本
    将流动相中的蛋白质样品溶解在1mg/ml(用于测定Ca 2+ /传感器蛋白的Rs中的Ca 2+ /依赖性变化),加入5mM EGTA或5mM CaCl 2 样品)


该议定书由Uversky先前发表的研究改编自1993年; Sorensen和Shea,1996; Astegno等,2016; Vallone等人,2016年。


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引用:La Verde, V., Dominici, P. and Astegno, A. (2017). Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography. Bio-protocol 7(8): e2230. DOI: 10.21769/BioProtoc.2230.