Gel Filtration Chromatography Protocol

Introduction

Gel-filtration chromatography, also known as size exclusion chromatography, is a versatile technique that permits the separation of proteins and other biological molecules. The gel filtration chromatography separates the proteins solely based on molecular size differences. For this, a porous matrix is used to which the molecules, for steric reasons, have different degrees of access. The matrix is enclosed in a chromatographic column, and the separation is accomplished by passing an aqueous buffer through the column. The molecules, confined outside the matrix beads, sweeps through the column by the mobile phase. An in-line UV monitor detects the separated protein zones and the fractions of the sample are collected for subsequent specific analysis. The gel-filtration chromatography has numerous applications including the fractionation of proteins and other water-soluble polymers, size determination and analysis, desalting, and buffer exchange.

Principle

The gel filtration chromatography is based on the molecular size and the hydrodynamic volume of the components. The molecules are separated by the differential exclusion or inclusion of solutes as they pass through the stationary phase containing heterosporous cross-linked polymeric gel or beads. Different permeation rates of the solute molecules cause them to sift in the interior of the gel particles. A column of the porous matrix is in equilibrium with the mobile phase for the separation of the molecules. Large molecules are entirely excluded from the pores and come first in the effluent. Smaller molecules get distributed between the mobile phase and the outside of the sieve. Then, they pass through the column at a slower rate and appear later in the effluent.

Apparatus

The gel filtration medium is enclosed in a column making a packed bed. The medium consists of a porous matrix selected on the basis of inertness and physical and chemical properties. The pores of the matrix are filled with the buffer equilibrated with the packed bed. The liquid inside the pores is referred to as the stationary phase and the liquid outside as the mobile phase. The signals from the column are taken by the detector, and the calculations are made.

Protocol (Irvine, 2001)

Group separation (desalting)

Gel preparation

1. Add calculated amount of dry [amazon link=”B00KIRS1G0″ link_icon=”amazon” /] to a volume of gel-filtration desalting buffer equal to twice the final gel volume.
2. Carefully stir the solution with a glass rod. Let the gel swell overnight at room temperature or for 3 hours in a 90°C water bath.
3. Allow the gel bed to settle and decant the solution to remove fines and broken beads. Repeat the decantation (4 or 5 times if necessary) and then dilute to get the final slurry with 50% (v/v) settled gel and 50% (v/v) GF buffer.

Pack column

4. Ensure that the column is clean and check the nets for any damage.
5. Mount the column vertically on a stable laboratory stand. Equip the column with an extension to hold the complete volume of the gel slurry.
6. Inject the gel-filtration (GF) buffer in outlet tubing until the support net is covered with 0.5 cm of the buffer.
7. Inject GF buffer into the inlet tubing of the adaptor until the net is wetted.
8. Swirl the gel slurry from step 3 and pour it into the column. Fill the remaining space with GF buffer. And, put lid on the column extension (or put the top adaptor on column).
9. Fill the buffer reservoir with GF buffer. Connect the reservoir to the pump with the help of a large tube.
10. Purge the pump with GF buffer and attach the outlet from the pump to the inlet of the column. Open the column outlet and start the pump at the flow rate. Continue the flow until the height of the gel bed becomes constant.
11. Turn the pump off, close the column outlet, remove the extension, adjust the bed height, and adjust the inlet adaptor.
12. Reattach the column to pump, open the column outlet, and resume flow conditions used in step 10 for 1 hour to stabilize the bed height.
13. Inspect the packed bed visually for cracks, trapped air, and particle aggregates. Determine the zones produced on the chromatogram.

Prepare and test the system

14. Calculate the amount of GF buffer necessary for the run and filter it plus a 50% excess through a 0.22-μm filter.
15. Assemble the GF system, placing the detector and recorder in line but leaving the column offline. Attach the buffer reservoir to pump and purge it with GF buffer.
16. Connect the outlet of the pump to the column via the injection valve, and run the system with GF buffer at a flow-rate set for separation.
17. Collect fractions with the fraction collector, and note the actual flow rate of the pump.

Determine the separating volume

18. Determine the void volume (Vo) by running a void marker and obtaining the elution volume.
19. Ascertain the total liquid volume (Vt) by running a total liquid volume marker and obtaining the elution volume.
20. Calculate the separating volume of the column (Vi) by subtracting the void volume from the total volume (Vt – Vo).

Chromatograph the sample

21. Dissolve the sample to be desalted in a gel-filtration buffer. Filter it through a 0.22-μm protein-compatible filter.
22. Open the outlet from the column, start the pump, and let two-bed volumes of the buffer pass through the column. Turn on the detector and stabilize the baseline.
23. Load sample applicator with the required volume of sample for desalting and switch the sample application valve to the load position.
24. Pass the buffer through the system at the appropriate flow rate and collect the fractions. Construct a chromatogram and calculate the elution volume as the time from the apex of the peak for the protein multiplied by the flow rate.
25. Wash the column with ≥1 column volume of the buffer containing an antibacterial agent. Close the column outlet and store it.

Protein fractionation

Column preparation

1. Prepare gel filtration matrix as indicated above except using the gel-filtration (GF) fractionation buffer wherever GF buffer is indicated.
2. Pack the column following steps 4-13 mentioned above.
3. Check the quality of the column (see step 14).
4. Assemble and test the system by following steps 15-17.

Evaluate the column

5. Chromatograph a colored marker (2 mg/ml Blue Dextran 2000 or 0.2 mg/ml vitamin B12 following steps 20 to 23) and determine the zones produced.
6. Chromatograph a low-molecular-weight marker (e.g., 5 mg/ml acetone following steps 20 to 23) and determine the column efficiency by constructing the chromatogram.
7. Calculate the number of theoretical plates per column using the equation N = 5.54(Vr/Wh)2 (where N = number of plates per column, Vr = elution volume of a peak, and Wh = width of the peak at half peak height).
8. Calculate the asymmetry factor of the peak according to the equation As = (b/a) (where a is the width of the leading part and b is the width of the tailing part of the peak).
9. Compare the calculated plate number and asymmetry factor for the column with the acceptance limits for these parameters.

Chromatography

10. Dissolve, apply, and chromatograph the protein sample to be fractionated.
11. Evaluate the collected fractions for purity.

Molecular size determination

1. Prepare the gel, pack the column, then assemble and test the GF system following steps 1-16 using GF fractionation buffer in place of the GF buffer.
2. Determine the void volume (Vo) and the total liquid volume (Vt) (follow steps 17 and 18).
3. Calibrate the column and check the flow rate during calibration by sampling effluent and weighing fractions.
4. Apply, elute, and chromatograph the sample.
5. Calculate the molecular size of the sample components using the calibration graph.

Applications

Assessment of the anticoagulant properties of heparin (Andersson. et al., 1976)

Heparin is an essential anti-coagulation protein that is activated by antithrombin III. Heparin could also affect coagulation and is critical for the studies regarding coagulation and anti-coagulation processes. In addition to thrombin, it inhibits the activated forms of other coagulation factors such as IX, X, and XI. In the study, a sample of heparin was fractionated using affinity chromatography on matrix-bound antithrombin III. The obtained fractions and sub-fractions were separated by gel filtration based on their molecular weights. It was found that about one-third of the heparin was bound to antithrombin and this fraction is responsible for 85% of the total anti-coagulation activity, measured in terms of thrombin inactivation. The results suggested that the antithrombin in the presence of heparin blocks several stages in the coagulation cascade. The fractionation of protein by gel-filtration chromatography enables the assessment of protein cascades involved in physiological processes.

Measurement of the plant cell wall permeability (Tepfer. & Taylor., 1981)

The permeability of the plant cell wall determines the ability of enzymes, polysaccharides, and extracellular glycoproteins to penetrate and alter the cell wall. The cell wall permeability could limit the macromolecules to alter the biochemical and physical properties of the wall. Gel-filtration chromatography enabled the detection of permeation of macromolecules into the cell wall matrix even at much lower concentrations. Using proteins of known size, a column packed with isolated cell wall fragments was calibrated, and the degree of protein penetration was measured.  The results showed that the proteins having a molecular weight, of 40,000 to 60,000 could penetrate a substantial portion of the cell wall space. Gel-filtration chromatography is a powerful separation technique for the estimation of plant cell wall permeability.

Proteomic characterization of human plasma high-density lipoprotein (Gordon., Deng., Lu., & Davidson., 2010)

The plasma levels of high-density lipoprotein (HDL) cholesterol are crucial to the incidence of cardiovascular disease. The modern proteomic technologies have identified 50 distinct proteins associated with HDL particles implicating the role of HDL in non-lipid transport processes. High-resolution size exclusion chromatography was used to fractionate normal human plasma to 17 phospholipid-containing sub-fractions. Then, the proteins were identified using a phospholipid-binding resin by electrospray ionization mass spectrometry. The identified proteins along with the HDL were found to be involved in complement regulation and protease inhibition. The technique allowed the visualization of HDL protein distribution across particle size with a higher resolution.

Analysis of protein biotherapeutics and their aggregates (Hong., Koza., & Bouvier., 2012)

Gel-filtration chromatography has been widely used not only to purify proteins but also to determine the sizes and relative distribution of macromolecules. The size-exclusion chromatography (SEC) is mainly used for routine and validated analyses because of the speed and reproducibility of the technique. The introduction of biologic-based therapeutics and associated protein aggregates have also been studied using the SEC. It has enabled the quantitation of dimers, trimers, and higher-order aggregates for biologic-based therapies including insulin, recombinant human growth hormone, and monoclonal antibodies. The size-exclusion chromatography has been found the most accurate technique for the analysis of proteins, aggregates, and biotherapeutics.

Precautions

• The separation process is based on the particle sizes so any damage in the particle could lead to false results.
• The buffers and the matrix should be degassed as air bubbles could lead to poor resolution.
• Preparation of the gel from thin suspension and column packing in stages could result in a poorly packed column.
• Avoid disturbing the bed as an uneven bed surface may lead to uneven separation.
• Maintain the experimental setup with proper care.

Strengths and limitations

• Gel-filtration chromatography is generally used to separate organic molecules and to determine their molecular weights and molecular weight distributions.
• Gel-filtration chromatography is an excellent technique for removing low-molecular-size contaminants from a purified protein sample for structural and functional analysis.
• The process can be conducted under mild conditions: from 37^oC to cold room temperature.
• The molecules can be separated with a high resolution and greater efficiency.
• The absence of a molecule-matrix binding prevents unnecessary damage to fragile molecules.
• Proteolysis is an issue while separating the proteins by gel-filtration chromatography.
• Because of the large size of the columns, large volumes of eluent are required which may lead to excessive running costs.

References

1. Irvine, B. G. (2001). Determination of molecular size by size-exclusion chromatography (gel filtration). Curr Protoc Cell Biol, Chapter 5: Unit 5.5.
2. S. Gordon., J. Deng., J. L. Lu., & Davidson., S. W. (2010). Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography. J Proteome Res, 9(10), 5239-49.
3. Tepfer., & Taylor., E. I. (1981). The permeability of plant cell walls as measured by gel filtration chromatography. Science, 213(4509), 761-3.
4. L. Andersson., W. T. Barrowcliffe., E. Holmer., A. E. Johnson., & Sims., G. E. (1976). Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin iii and by gel filtration. Thromb Res, 9(6), 575-83.
5. Hong., S. Koza., & Bouvier., S. E. (2012). Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates. J Liq Chromatogr Relat Technol, 35(20), 2923-2950.