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Molecular Techniques and Methods

Protein Purification by Gel Filtration Chromatography

INTRODUCTION

Gel filtration chromatography separates proteins in order of large to small molecules. Protein mixtures are applied to a gel-filtration (GF) column containing a chromatographic matrix of defined pore size. Proteins are eluted with an aqueous buffer, collected as individual chromatographic fractions, and analyzed separately. Gel filtration can be used to separate proteins based on differences in their molecular size, or to desalt proteins (i.e., remove low-molecular-weight contaminants such as salts, amino acids, and peptides, etc.). A separation of the various proteins in a sample results from differences in their abilities to enter the pores. In general, large proteins will not enter the gel pores and will elute rapidly from the column in the void volume. Smaller proteins will repeatedly enter and leave the pores of the gel and therefore will remain longer in the column. Hence, proteins will elute in order of decreasing molecular size.

MATERIALS AND SOLUTIONS

Commercially Available Gel Filtration (GF) Gel Matrix

Gel Type	Fractionation Range (molecular weight)
Sephadex G-10	<700
Sephadex G-25	1,000-5,000
Bio-Gel P-60	3,000-60,000
Sephadex G-75	3,000-70,000
Sephadex G-100	4,000-100,000
Bio-Gel P-100	5,000-100,000
Sephadex G-200	5,000-250,000
Bio-Gel P-200	30,000-200,000
Sephacryl S-300	10,000-1,500,000
Sepharose 2B	70,000-40,000,000

Gel Filtration (GF) Buffer
Tris-HCl buffer, Sodium phosphate buffer, and Sodium acetate buffer at various pHs are most commonly used. An ionic strength of at least 0.05 M is recommended to reduce nonspecific interactions between the proteins being separated and the chromatographic matrix.

Gel Filtration (GF) Chromatography Column

Carpenter's level Column Extension or Gel Reservoir

Peristaltic Pump

UV Detector

Fraction Collector

PROCEDURES

Swelling the Gel

1. Swell the GF (Gel Filtration) gel matrix in an appropriate GF buffer.
Allow to swell completely.

2. After allowing the gel to settle, aspirate or decant the fine gel particles which have not settled to the gel bed.

3. Resuspend the settled gel in an equal volume of GF buffer to form a thick suspension, pour the slurry into a filtration flask, degas the gel in order to remove trapped air.

4. The volume of swelling buffer and the swelling time required for a given gel depends on the gel type. The dry gel should be gradually added to the buffer, while gently stirring the suspension with a glass rod.
Note: Do not stir with a magnetic stirrer, since the gel beads will be pulverized.

5. Suspend the gel in twice the approximate bed volume (i.e., milliliters of swollen gel).
Gels may be rapidly swollen by heating the slurry at 90^oC for 5 hours, using a water bath to control the temperature. Swelling at room temperature can be considerably slower, especially for large pore gels.

6. If a GF gel matrix is purchased preswollen, it should be washed with excess buffer on a Buchner funnel to remove antimicrobial agents contained in the storage buffer. Resuspend the settled gel in GF buffer, degas the suspension.

Filling the Column

7. Mount the column vertically on a laboratory stand. A carpenter's level should be used to determine that the column is vertical.

8. Using a syringe, inject GF buffer into the column outlet tubing until the empty column is filled with buffer to just above the bed support screen. Leave the syringe in place to block the end of the outlet tubing.

(This procedure removes trapped air from below the support screen.)

9. Pour an appropriate volume of the gel suspension in order to fill completely the column to the required column bed height. The gel suspension should be poured onto a glass rod whose end touches the inner column wall. This will result in a smooth flow of the gel suspension without unnecessary turbulence and introduction of air.

10. Since the volume of the slurry usually exceeds the desired column bed volume, it is convenient to use a gel reservoir or column extension to hold the excess slurry.

11. After the column is packed to the desired bed height, carefully pipet a 1 cm layer of buffer onto the top of the gel. After completely filling the inlet tubing and end fitting with buffer, connect the end fitting to the column.

Packing and Washing the Column

12. Before removing the syringe from the outlet tubing, adjust the buffer reservoir to provide an appropriate operating pressure or use a peristaltic pump to control the flow rate.

For nonrigid gels (e.g., Sephadex G-75, G- 100, and G-200 or Sepharose), operating pressures should not exceed the manufacturer defined limits.

For rigid gel (e.g., Sephadex G-10 to G-50 or Sephacryl), packing can be carried out at higher flow rates using a peristaltic pump.

13. Remove the syringe from the column outlet tubing and start the flow. Wash the column with 2 to 3 bed volumes of buffer in order to pack the bed and to equilibrate the column with buffer. A slightly higher flow rate can be used for packing than will be used for chromatographic separation.

14. Close the outlet tubing. Inspect the packed bed, illuminating from behind by a flashlight to detect cracks or trapped air in the column bed.

Determining the Column Void Volume

15. Close the outlet tubing and remove most of the buffer from above the column bed by aspiration. Open the Outlet tubing and allow the remaining buffer to penetrate into the gel, but be careful not to let the center of the gel become dry. Close the outlet tubing.

16. Using a pasteur pipet, apply a sample of protein (0.2 to 0.5 mg/ml), whose molecular weight is known to be greater than the void volume of the GF matrix being used for the separation, in a buffer volume equal to 1% of the total bed volume of the column is layered on top of gel.

17. Open the column outlet tubing and allow the sample to penetrate into bed.

18. Wash the remaining sample from the column wall by applying small amounts of buffer from a Pasteur pipet.

19. After the sample is applied, carefully pipet a 1 cm layer of buffer onto the top of the gel, and after completely filling the inlet tubing and end fitting with buffer, connect the end fitting to the column.

20. Allow the elution to proceed at an appropriate hydrostatic pressure or flow rate.

21. Collect the column effluent using an automated fraction collector. It is convenient to collect 100 fractions each containing a volume equivalent to approximately 1 % of the total bed volume.

22. Measure the A₂₈₀ of each fraction. Calculate the void volume by multiplying the volume collected in the individual fractions by die number of the fraction containing the maximal UV-absorbing protein. The void volume should be approximately 1/3 of the total bed volume.

Dissolving and Applying Sample

23. Dissolve the sample containing a mixture of proteins in the buffer used in the gel filtration column. For maximum resolution, the sample volume should be 1-5% of the column bed volume.

24. In order to optimize chromatographic peak shape, it is necessary that the viscosity of the sample solution be no greater than twice that of the elution buffer.

Collecting the Column Fractions

25. Measure the A₂₈₀ of the individual chromatographic fractions or the column effluent in order to detect proteins. Proteins containing tyrosine and tryptophan residues will absorb at 280 nm.

NOTES

The resolution of two protein bands during gel filtration is related to the square root of column length, particle size of gel, flow rate of buffer, and fractionation range of gel. A protein separation is best carried out on a long column (e.g., > 50 cm) with an internal diameter between 1 and 2.5 cm.

For most protein separations, fine-sized particles should be used, since both chromatographic resolution and flow rate will remain high.
For very large-scale protein separations, medium-sized particles are recommended.
For the highest resolution, the linear flow rate should be maintained between 2 and 10 cm/hour. The corresponding column flow rate in ml/hour can be calculated by multiplying linear flow rate by the cross sectional area of the column (cm²).

The choice of a gel-filtration matrix depends on the molecular size of the protein being purified, as well as the molecular sizes of contaminants. It is important that the molecular size of the protein being separated lie within the middle of the fractionation range of the column matrix used for the separation.

The ionic strength of buffers used for gel filtration should be 0.05 M or greater, and the pH of the buffer should not exceed the operating pH range prescribed by the manufacturer for a chromatographic matrix.

The protein must be soluble in the buffer chosen. If necessary, the protein can be dissolved in buffers containing chaotropic agents (e.g., 6 M guanidine-HCl and 8 M urea), organic solvents at low concentrations, and detergents, and the separation may be carried out in such buffers if the chosen gel matrix is stable under these conditions.

Protein Molecular Weight Standard.

Protein	Molecular Weight
Cytochrome c	11,700
Myoglobin	16,800
Trypsinogen	24,000
Carbonic anhydrase	29,000
Ovalbumin	45,000
Hemoglobin	64,500
Bovine serum albumin	66,000
Transferrin	74,000
Immunoglobulin G	158,000
Fibrinogen	341,000
Ferritin	470,000
Thyroglobulin	670,000

KIT INFORMATION

REFERENCES

Andrews, P. 1970. Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods of Biochemical Analysis (Glikc, D., ed.). Vol. 18, pp.1-53. lnterscience, New York.

Fischer, L. 1980. Gel-filtration Chromatography. Elsevier, Amsterdam.

Porath. J. and Flodin, P. 1959. Gel filtration: A method for desalting and group separation. Nature 183:1657-1659.

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