GEL FILTRATION AND ION EXCHANGE CHROMATOGRAPHY
GEL FILTRATION AND ION EXCHANGE CHROMATOGRAPHY
Background
Ion exchange chromatography
Ion exchange chromatography separates substances on the basis of their charge. There are two general classes of ion exchange media or resins; anion-exchange media, which have positively charged groups attached to the media, and bind to anionic (negatively charged) compounds, and cation-exchange media, which have negatively charged groups attached to the media, and bind to cationic compounds. Amberlite CG-50 is a cation-exchange resin, i.e., it has covalently attached carboxylate groups which, at neutral pH, are charged-balanced by associated sodium ions. Proteins with significant regions of opposing charge will displace the sodium and bind to this column material by ionic attraction. The initial protein binding is usually done in a solution of low ionic strength where the protein, in this case cytochrome c, can displace the sodium ions, and bind to the column material. A solution containing the desired protein is passed through a column of ion exchange media, and the eluant checked to ensure that the protein has bound to the column. The column is then washed with 2 - 5 column volumes of low ionic strength buffer to remove any unbound material. At this point, the protein bound to the column can be selectively released by increasing the salt concentration. The salt competes with the protein for the charged groups on the column, and at a salt concentration characteristic for each protein, the protein is eluted. In this experiment, cytochrome c is eluted with 0.5 M NaCl, and we used a "step-wise" increase in concentration from 0 to 0.5 M NaCl to elute it. As the high-salt buffer moves through the column, the cytochrome c is displaced from the column material by the sodium ions and collects as a red line at the interface between the initial, low-salt buffer and the high-salt buffer with which it is eluted. Alternative elution methods include using a salt concentration gradient (most selective) to elute the protein, or a "batch" purification procedure, where the column material is mixed with the protein solution before pouring the column. Salt gradients generally give the best purification, while batch procedures are less efficient but sometimes more convenient. Gel filtration chromatography
Gel filtration chromatography separates substances on the basis of their size. "Sephadex," one of the first gel filtration media, is prepared by cross linking dextrans, which are carbohydrate polymers, to produce beads containing an extensive network of channels or pores. If a sample containing a mixture of compounds is applied to a column filled with Sephadex beads, the compounds that are too large to enter the pores are excluded from the interior of the beads and so move down the column rapidly. Conversely, those compounds small enough to enter inside the pores have a much longer potential path through the column and so travel down the column more slowly. The large number of hydroxyl groups renders the gel extremely hydrophilic, and provides a non denaturing environment for most proteins. The G-types of Sephadex differ in their degree of cross linking, and hence in their degree of swelling when hydrated, and in their fractionation range. Sephadex G-50, for example, separates globular proteins in the 1500-30,000 molecular weight range.
2. Obtain the column material from the instructor. Make sure that the column and column material are at room temperature. Temperature gradients will make the column pack and flow unevenly, preventing good separations.
3. Assemble the columns, and fill about 1/4 full of buffer (see the demonstration column in the lab).
4. Suspend the column material in excess buffer by swirling, and pour it into the column until the column is completely full.
5. Open the valve at the bottom of the column.
6. Allow the column material to settle. Add additional column material until the packed bed is the desired height (~10cm). Do not let the column dry out in between each addition.
7. Add buffer, making sure that new buffer (20 mM sodium phosphate, pH 8.0) is added frequently enough to keep clear solution above the top to the column material.
8. Allow at least two column volumes of buffer to pass through the column. Fill the top part of the column with buffer, cap it and close the valve at the bottom.
Sample Application & Elution
Ion exchange chromatography
For ion exchange chromatography, the sample solution volume can be large, and the solution added simply by using it to replace the column buffer. However the sample solution must not prevent the protein from binding to the column material.
1. Apply the sample by replacing the column buffer with the sample solution and allowing the solution to flow through the column. This can be done "automatically" by dipping the piece of tubing plugged into the cap of the column directly into the sample bottle. Initiate the flow using a syringe to pull the liquid into the tubing and place the sample solution bottle as high as possible above the level of the column to maintain the flow rate (see demonstration in lab).
2. After applying the sample, wash the column with several (2 - 5) column volumes of buffer to remove any unbound or weakly bound proteins.
3. Allow the buffer to drain down exactly to the top of the column material.
4. Elute the protein by carefully adding the high salt buffer to the top of the column. Add about 1 mL/cm2 of column surface. Touch the sample applicator to the wall of the column as you add the buffer to keep from disturbing the top of the column bed.
5. Allow the high salt buffer to drain into the column, but do not let any column material dry.
6. Add another volume of high salt buffer, then keep the column filled with buffer, being careful to not disturb the top of the column material.
7. Collect the eluant in several small fractions (~0.5 mL).
Gel filtration chromatography
For gel filtration chromatography, it is important to apply the sample to the top of the column evenly, in as small a volume as possible. This will prevent excessive dilution and poor separation.
1. Allow the buffer to drain down exactly to the top of the column material.
2. Carefully add about 1 mL of the sample/cm2 of column bed to the top of the column. Touch the sample applicator to the wall of the column as you add the sample to keep from disturbing the top of the column bed.
3. Allow the sample to drain into the column, but do not let any column material dry. Carefully add a volume of buffer equal to the sample volume, using the sample applicator and start collecting .
4. Still collecting, add another volume of buffer, then fill the column with buffer, being careful to not disturb the top of the column material.
5. Collect about 30 fractions (~0.5 mL)
Sample Collection
Sample elution can be monitored by color, if it is a colored protein, (hence chromatography) or by its UV absorbance for colorless proteins. UV-detectors and fraction collectors are frequently useful for slow columns and/or colorless proteins. By choosing a colored protein and a short, fast column we just avoided these difficulties.
1. Select the fractions that contain cytochrome c on the basis of their color (anywhere from red to pinkish).
2. Pool the fractions for which the color is the most pronounced, and use the least colored ones (from the selection made in step1 ) to rinse all the tubes, in order to maximize the amount of cytochrome c collected.
3. Store the cytochrome c solution frozen at -20°C, with your name, the date, and sample name written on the container.
Background
Ion exchange chromatography
Ion exchange chromatography separates substances on the basis of their charge. There are two general classes of ion exchange media or resins; anion-exchange media, which have positively charged groups attached to the media, and bind to anionic (negatively charged) compounds, and cation-exchange media, which have negatively charged groups attached to the media, and bind to cationic compounds. Amberlite CG-50 is a cation-exchange resin, i.e., it has covalently attached carboxylate groups which, at neutral pH, are charged-balanced by associated sodium ions. Proteins with significant regions of opposing charge will displace the sodium and bind to this column material by ionic attraction. The initial protein binding is usually done in a solution of low ionic strength where the protein, in this case cytochrome c, can displace the sodium ions, and bind to the column material. A solution containing the desired protein is passed through a column of ion exchange media, and the eluant checked to ensure that the protein has bound to the column. The column is then washed with 2 - 5 column volumes of low ionic strength buffer to remove any unbound material. At this point, the protein bound to the column can be selectively released by increasing the salt concentration. The salt competes with the protein for the charged groups on the column, and at a salt concentration characteristic for each protein, the protein is eluted. In this experiment, cytochrome c is eluted with 0.5 M NaCl, and we used a "step-wise" increase in concentration from 0 to 0.5 M NaCl to elute it. As the high-salt buffer moves through the column, the cytochrome c is displaced from the column material by the sodium ions and collects as a red line at the interface between the initial, low-salt buffer and the high-salt buffer with which it is eluted. Alternative elution methods include using a salt concentration gradient (most selective) to elute the protein, or a "batch" purification procedure, where the column material is mixed with the protein solution before pouring the column. Salt gradients generally give the best purification, while batch procedures are less efficient but sometimes more convenient. Gel filtration chromatography
Gel filtration chromatography separates substances on the basis of their size. "Sephadex," one of the first gel filtration media, is prepared by cross linking dextrans, which are carbohydrate polymers, to produce beads containing an extensive network of channels or pores. If a sample containing a mixture of compounds is applied to a column filled with Sephadex beads, the compounds that are too large to enter the pores are excluded from the interior of the beads and so move down the column rapidly. Conversely, those compounds small enough to enter inside the pores have a much longer potential path through the column and so travel down the column more slowly. The large number of hydroxyl groups renders the gel extremely hydrophilic, and provides a non denaturing environment for most proteins. The G-types of Sephadex differ in their degree of cross linking, and hence in their degree of swelling when hydrated, and in their fractionation range. Sephadex G-50, for example, separates globular proteins in the 1500-30,000 molecular weight range.
Column Preparation
Reagents- 250 mL - 200 mM sodium phosphate, pH 8.0 (10X stock)
1 L - 20 mM sodium phosphate, pH 8.0 (buffer)
100 mL - 20 mM sodium phosphate, 0.5 M NaCl, pH 8.0 (high salt buffer)
~5 mL hydrated volume/column Amberlite CG-50 weakly acidic ion exchange resin (5 mL/g dry wt; equilibrate with 8 vol. 2N NaOH, then buffer until pH ~ 8).
| Buffer | pH | Volume |
| sodium phosphate 200 mM | 8.0 | 250 mL |
| sodium phosphate 20 mM | 8.0 | 1 L |
| sodium phosphate 20 mM, NaCl 0.5 M (high salt buffer) | 8.0 | 100 mL |
3. Assemble the columns, and fill about 1/4 full of buffer (see the demonstration column in the lab).
4. Suspend the column material in excess buffer by swirling, and pour it into the column until the column is completely full.
5. Open the valve at the bottom of the column.
6. Allow the column material to settle. Add additional column material until the packed bed is the desired height (~10cm). Do not let the column dry out in between each addition.
7. Add buffer, making sure that new buffer (20 mM sodium phosphate, pH 8.0) is added frequently enough to keep clear solution above the top to the column material.
8. Allow at least two column volumes of buffer to pass through the column. Fill the top part of the column with buffer, cap it and close the valve at the bottom.
Sample Application & Elution
Ion exchange chromatography
For ion exchange chromatography, the sample solution volume can be large, and the solution added simply by using it to replace the column buffer. However the sample solution must not prevent the protein from binding to the column material.
1. Apply the sample by replacing the column buffer with the sample solution and allowing the solution to flow through the column. This can be done "automatically" by dipping the piece of tubing plugged into the cap of the column directly into the sample bottle. Initiate the flow using a syringe to pull the liquid into the tubing and place the sample solution bottle as high as possible above the level of the column to maintain the flow rate (see demonstration in lab).
2. After applying the sample, wash the column with several (2 - 5) column volumes of buffer to remove any unbound or weakly bound proteins.
3. Allow the buffer to drain down exactly to the top of the column material.
4. Elute the protein by carefully adding the high salt buffer to the top of the column. Add about 1 mL/cm2 of column surface. Touch the sample applicator to the wall of the column as you add the buffer to keep from disturbing the top of the column bed.
5. Allow the high salt buffer to drain into the column, but do not let any column material dry.
6. Add another volume of high salt buffer, then keep the column filled with buffer, being careful to not disturb the top of the column material.
7. Collect the eluant in several small fractions (~0.5 mL).
Gel filtration chromatography
For gel filtration chromatography, it is important to apply the sample to the top of the column evenly, in as small a volume as possible. This will prevent excessive dilution and poor separation.
1. Allow the buffer to drain down exactly to the top of the column material.
2. Carefully add about 1 mL of the sample/cm2 of column bed to the top of the column. Touch the sample applicator to the wall of the column as you add the sample to keep from disturbing the top of the column bed.
3. Allow the sample to drain into the column, but do not let any column material dry. Carefully add a volume of buffer equal to the sample volume, using the sample applicator and start collecting .
4. Still collecting, add another volume of buffer, then fill the column with buffer, being careful to not disturb the top of the column material.
5. Collect about 30 fractions (~0.5 mL)
Sample Collection
Sample elution can be monitored by color, if it is a colored protein, (hence chromatography) or by its UV absorbance for colorless proteins. UV-detectors and fraction collectors are frequently useful for slow columns and/or colorless proteins. By choosing a colored protein and a short, fast column we just avoided these difficulties.
1. Select the fractions that contain cytochrome c on the basis of their color (anywhere from red to pinkish).
2. Pool the fractions for which the color is the most pronounced, and use the least colored ones (from the selection made in step1 ) to rinse all the tubes, in order to maximize the amount of cytochrome c collected.
3. Store the cytochrome c solution frozen at -20°C, with your name, the date, and sample name written on the container.
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