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Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins
CHAPTER NINE
Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins Krisna C. Duong-Ly, Sandra B. Gabelli1 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Standardization of the Gel Filtration Column 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 5.5 Tip 5.6 Tip 5.7 Tip 6. Step 2 Determination of the Sizes of Protein Species in a Sample 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip 6.6 Tip 6.7 Tip References
106 108 108 109 109 109 110 110 110 110 111 111 111 111 111 112 112 112 113 113 113 113 113 114
Abstract The protocol described here allows the student to construct a standard curve for a gel filtration column with a separation range of 5–250 kD. The size (hydrodynamic radius) of a protein species stable in a buffer containing Tris–HCl, NaCl, and DTT is determined Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00009-4
#
2014 Elsevier Inc. All rights reserved.
105
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Krisna C. Duong-Ly and Sandra B. Gabelli
using this column. Modifications may be made to the buffer to accommodate the protein of interest and the separation range of the column.
1. THEORY Gel filtration chromatography, also known as size exclusion chromatography, is used to separate molecules of different sizes. In addition to separating different proteins of varying size, one may resolve oligomeric forms of a particular protein. Furthermore, this technique can be used to exchange the buffer of a sample for a different one. This specific type of column is commonly referred to as a desalting column. Gel filtration columns consist of a matrix of beads that contain sieves of a particular size. The beads of gel filtration columns consist of cross-linked polyacrylamide, agarose, dextran, or a combination of any of these (Scopes, 1993). Large molecules such as proteins and polymers may or may not enter the beads, depending on their sizes, and will elute before small compounds such as ions and buffer salts, which can enter the sieves in the matrix of the stationary phase. Note that the word ‘size’ in this discussion correlates with the Stokes radius of the molecule, which takes into account the interaction between molecules and water (Scopes, 1993). In desalting columns, the sieves are so small, only small salts and buffer salts can enter them, and proteins will elute in the buffer used for the column. Size exclusion columns are characterized by their cutoff size, void volume, and column volume (Fig. 9.1). Cutoff size refers to the approximate size of the largest molecule that may enter the beads. For simplicity, this figure is often given as a molecular weight rather than specific dimensions, so interpret this cautiously if the protein of interest is any shape other than globular. Any species larger than this size will not interact with the beads and will elute in the void volume, the volume of the column that is not occupied by the matrix. The column volume refers to the total accessible volume of the column to solvent. Small compounds present in the sample, such as salts, will enter the matrix and will elute at approximately the column volume. If beads are spherical, the void volume will equal 25–30% of the column volume (Scopes, 1993). While the manufacturer’s information often gives the cutoff size for a particular gel filtration column, the column volume and void volume should be determined before an experiment is performed. Also, the elution volumes of proteins in the size range of interest should be determined. There is a
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Figure 9.1 Theoretical gel filtration chromatograph of a sample. In this case, a small amount of aggregated protein is larger than the cutoff size for the column and elutes at the void volume. The bulk of the protein elutes within the separation range of the column and is separated from the aggregates. The protein sample was originally in a buffer of higher conductivity than that of the column and excess salts eluted at the column volume.
linear relationship between log10 of the size of a particular protein and the ratio of the elution volume of that protein to the void volume (Whitaker, 1963). The size is approximated using the molecular weight of the protein species. Before a new column is used, a standard curve should be made using proteins of known molecular weights. Several kits with various proteins are available commercially. A high molecular weight species such as blue dextran (molecular weight 2000 kD) can be used to determine the void volume of the column (Cutler, 1998). Unlike other chromatographic methods, there is no binding between the stationary phase and elements of the mobile phase. One must be wary of the volume of sample injected; if the volume of the sample is large, the resolution of separation will be low. A good ‘rule of thumb’ is to inject less than 3% of the column volume (Scopes, 1993). Also, the eluted proteins are more dilute than the original sample and dilution increases if the protein elutes at larger elution volumes. Higher resolution can be obtained by using longer columns. A larger volume of sample can be loaded onto the column by increasing the circumference of the column. If packing a column, use care
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to pack the beads uniformly. Differences in bead density along the length of the column will affect the elution of different molecular weight species. Generally, gel filtration chromatography is used as a final purification step after at least one other purification step. This method should not be used as an initial protein purification step after cell lysis since there are too many proteins with similar sizes. Affinity chromatography (see Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Immunoaffinity purification of proteins, Strep-tagged protein purification, Proteolytic affinity tag cleavage or Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins) and ion exchange chromatography (see Using ion exchange chromatography to purify a recombinantly expressed protein) are more ideally suited for a first purification step. If the goal of the experiment is to separate different oligomeric species, the initial sample should be concentrated (see TCA Precipitation or Salting out of proteins using ammonium sulfate precipitation) to a very small volume to prevent overlapping elution of two different species. If there is not a reasonable amount of separation between species, one may concentrate the sample further or use a longer column. Ideally, this experiment should be performed using an FPLC system capable of monitoring the UV absorbance of the protein(s) of interest and a fraction collector.
2. EQUIPMENT FPLC system (with UV detector and fraction collector) Gel filtration column Vacuum filtration assembly (sidearm flask and filter holder) 0.22-mm filters (for filtration assembly) Syringes 0.22-mm syringe filters
3. MATERIALS Tris-hydrochloride (Tris-HCl) Sodium chloride (NaCl) Dithiothreitol (DTT) Potassium chloride (KCl)
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Gel Filtration Kit for Molecular Weights 12,000-200,000 (Sigma cat # MWGF200, adjust if necessary for column)
3.1. Solutions & buffers Step 1 Molecular weight standards buffer Component
Final concentration
Stock
Amount
Tris–HCl, pH 7.5
50 mM
1M
200 ml
KCl
100 mM
1M
400 ml
Add water to 4 l and pass through a 0.22-mm filter
Step 2 Elution buffer Component
Final concentration
Stock
Amount
Tris–HCl, pH 7.5
50 mM
1M
50 ml
NaCl
150 mM
4M
37.5 ml
DTT
0.1 mM
1M
0.1 ml
Add water to 1 l and pass through a 0.22-mm filter
4. PROTOCOL 4.1. Preparation Obtain a reasonably pure protein sample (>90% pure) for characterization by gel filtration through another chromatography method, preferably ion exchange chromatography (see Using ion exchange chromatography to purify a recombinantly expressed protein) or affinity chromatography (see Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Immunoaffinity purification of proteins, Strep-tagged protein purification, Proteolytic affinity tag cleavage or Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins). If possible, concentrate the sample to 1 ml. If this is not possible, concentrate the sample to less than 3% of the column volume (see TCA Precipitation or Salting out of proteins using ammonium sulfate precipitation). Follow the column manufacturer’s instructions for placing the column in water. Follow the instructions of the gel filtration molecular
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Krisna C. Duong-Ly and Sandra B. Gabelli
Figure 9.2 Flowchart of the complete protocol, including preparation.
weight kit for dissolving and filtering the standardized proteins and the blue dextran. Prepare tubes for collecting fractions.
4.2. Duration Preparation
About 1–2 days
Protocol
Varies (based on column size)
See Fig. 9.2 for the flowchart of the complete protocol.
5. STEP 1 STANDARDIZATION OF THE GEL FILTRATION COLUMN 5.1. Overview Prepare the column for the protein standards and blue dextran and determine their elution volumes in order to make a standard curve.
5.2. Duration Variable (depends on column size) 1.1 Equilibrate the column with at least 2 column volumes of the molecular weight standards buffer. The flow rate should be 0.3–0.5% of the column volume per minute. Monitor the UV absorbance and when the reading is stable, set it to ‘0.’
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1.2 Inject 1 ml of the blue dextran solution and elute with 2 column volumes of the molecular weight standards buffer. Measure the elution volume of blue dextran to the center of the elution peak. This is the void volume. 1.3 Inject 1 ml of one of the protein standards and elute with 2 column volumes of the molecular weight standards buffer. Measure the elution volume. 1.4 Repeat Step 1.3 for the remaining protein standards. 1.5 Graph the log10 of the molecular weight of each protein standard versus the ratio of its elution volume to the void volume. Determine the line of best fit. This is the standard curve.
5.3. Tip If the UV absorbance does not stabilize after 2 column volumes, consider performing a rigorous cleaning of the column according to the manufacturer’s instructions or running more buffer over the column until the UV absorbance reading is stable.
5.4. Tip The elution volume includes the volume of sample that is injected; thus, one should set ‘0’ ml to be the time at which the injection of the sample begins.
5.5. Tip Eluting with 2 column volumes prepares the column for the next standard. Thus, a one column volume elution is sufficient for the final protein standard.
5.6. Tip This step may be skipped if the column has been standardized previously and has not suffered any changes due to repacking, etc.
5.7. Tip If a different molecular weight standards kit is used, use the buffer recommended for that kit. The buffer used in this protocol was recommended by Sigma for this particular product. See Fig. 9.3 for the flowchart of Step 1.
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Krisna C. Duong-Ly and Sandra B. Gabelli
Figure 9.3 Flowchart of Step 1.
6. STEP 2 DETERMINATION OF THE SIZES OF PROTEIN SPECIES IN A SAMPLE 6.1. Overview Purify a protein sample using gel filtration chromatography and determine the molecular weight of any species present.
6.2. Duration About 1 day 2.1 Remove the molecular weight standards buffer by washing the column with at least half a column volume of water. 2.2 Equilibrate the column with 2 column volumes of the elution buffer. When the UV absorbance reading is stable, set it to ‘0.’ 2.3 Pass the sample through a 0.22-mm syringe filter. 2.4 Inject the sample and elute with at least 1 column volume of the elution buffer. Measure the elution volume of each protein species. 2.5 Calculate the ratio of the elution volume to the void volume and use the standard curve to determine the size of the eluted species.
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6.3. Tip If the sample clogs the syringe filter, it may contain large aggregates. Use multiple syringe filters if this occurs.
6.4. Tip If the UV absorbance contains spikes or is unusually high and DTT is present, this indicates that the DTT may have oxidized. Make new buffer and add DTT right before the experiment. Avoid using buffers containing DTT that are more than 1-day old.
6.5. Tip If the column goes over pressure, decrease the flow rate. If this is a recurring problem, follow the column manufacturer’s instructions for rigorously cleaning the column.
6.6. Tip If the protein contains no Trp, it will not absorb appreciably at 280 nm. In this case, monitor the absorbance at 260 nm (for Tyr), or at another wavelength, to detect the protein.
6.7. Tip Salt (>150 mM ) is required in the protein and elution buffers to prevent protein adhesion to the beads. Additionally, if there is a difference between the conductivities
Figure 9.4 Flowchart of Step 2.
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of the sample and of the elution buffer, it will be apparent in the conductivity measurements at the position of the column volume. See Fig. 9.4 for the flowchart of Step 2.
REFERENCES Referenced Literature Scopes, R. (1993). Protein Purification: Principles and Practice (3rd ed.). New York: Springer. Whitaker, J. R. (1963). Analytical Chemistry, 35, 1950. Cutler, P. (1998). Size-exclusion chromatography. In R. Rapley & J. M. Walker (Eds.), Molecular Biomethods Handbook. (pp. 451–460): Humana Press.
Referenced Protocols in Methods Navigator Protein Affinity Purification using Intein/Chitin Binding Protein Tags. Purification of His-tagged proteins. Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag. Purification of GST-tagged proteins. Immunoaffinity purification of proteins. Strep-tagged protein purification. Proteolytic affinity tag cleavage. Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins. Using ion exchange chromatography to purify a recombinantly expressed protein. TCA Precipitation. Salting out of proteins using ammonium sulfate precipitation.
Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins Krisna C. Duong-Ly, Sandra B. Gabelli1 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Standardization of the Gel Filtration Column 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 5.5 Tip 5.6 Tip 5.7 Tip 6. Step 2 Determination of the Sizes of Protein Species in a Sample 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip 6.6 Tip 6.7 Tip References
106 108 108 109 109 109 110 110 110 110 111 111 111 111 111 112 112 112 113 113 113 113 113 114
Abstract The protocol described here allows the student to construct a standard curve for a gel filtration column with a separation range of 5–250 kD. The size (hydrodynamic radius) of a protein species stable in a buffer containing Tris–HCl, NaCl, and DTT is determined Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00009-4
#
2014 Elsevier Inc. All rights reserved.
105
106
Krisna C. Duong-Ly and Sandra B. Gabelli
using this column. Modifications may be made to the buffer to accommodate the protein of interest and the separation range of the column.
1. THEORY Gel filtration chromatography, also known as size exclusion chromatography, is used to separate molecules of different sizes. In addition to separating different proteins of varying size, one may resolve oligomeric forms of a particular protein. Furthermore, this technique can be used to exchange the buffer of a sample for a different one. This specific type of column is commonly referred to as a desalting column. Gel filtration columns consist of a matrix of beads that contain sieves of a particular size. The beads of gel filtration columns consist of cross-linked polyacrylamide, agarose, dextran, or a combination of any of these (Scopes, 1993). Large molecules such as proteins and polymers may or may not enter the beads, depending on their sizes, and will elute before small compounds such as ions and buffer salts, which can enter the sieves in the matrix of the stationary phase. Note that the word ‘size’ in this discussion correlates with the Stokes radius of the molecule, which takes into account the interaction between molecules and water (Scopes, 1993). In desalting columns, the sieves are so small, only small salts and buffer salts can enter them, and proteins will elute in the buffer used for the column. Size exclusion columns are characterized by their cutoff size, void volume, and column volume (Fig. 9.1). Cutoff size refers to the approximate size of the largest molecule that may enter the beads. For simplicity, this figure is often given as a molecular weight rather than specific dimensions, so interpret this cautiously if the protein of interest is any shape other than globular. Any species larger than this size will not interact with the beads and will elute in the void volume, the volume of the column that is not occupied by the matrix. The column volume refers to the total accessible volume of the column to solvent. Small compounds present in the sample, such as salts, will enter the matrix and will elute at approximately the column volume. If beads are spherical, the void volume will equal 25–30% of the column volume (Scopes, 1993). While the manufacturer’s information often gives the cutoff size for a particular gel filtration column, the column volume and void volume should be determined before an experiment is performed. Also, the elution volumes of proteins in the size range of interest should be determined. There is a
Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins
107
Figure 9.1 Theoretical gel filtration chromatograph of a sample. In this case, a small amount of aggregated protein is larger than the cutoff size for the column and elutes at the void volume. The bulk of the protein elutes within the separation range of the column and is separated from the aggregates. The protein sample was originally in a buffer of higher conductivity than that of the column and excess salts eluted at the column volume.
linear relationship between log10 of the size of a particular protein and the ratio of the elution volume of that protein to the void volume (Whitaker, 1963). The size is approximated using the molecular weight of the protein species. Before a new column is used, a standard curve should be made using proteins of known molecular weights. Several kits with various proteins are available commercially. A high molecular weight species such as blue dextran (molecular weight 2000 kD) can be used to determine the void volume of the column (Cutler, 1998). Unlike other chromatographic methods, there is no binding between the stationary phase and elements of the mobile phase. One must be wary of the volume of sample injected; if the volume of the sample is large, the resolution of separation will be low. A good ‘rule of thumb’ is to inject less than 3% of the column volume (Scopes, 1993). Also, the eluted proteins are more dilute than the original sample and dilution increases if the protein elutes at larger elution volumes. Higher resolution can be obtained by using longer columns. A larger volume of sample can be loaded onto the column by increasing the circumference of the column. If packing a column, use care
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Krisna C. Duong-Ly and Sandra B. Gabelli
to pack the beads uniformly. Differences in bead density along the length of the column will affect the elution of different molecular weight species. Generally, gel filtration chromatography is used as a final purification step after at least one other purification step. This method should not be used as an initial protein purification step after cell lysis since there are too many proteins with similar sizes. Affinity chromatography (see Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Immunoaffinity purification of proteins, Strep-tagged protein purification, Proteolytic affinity tag cleavage or Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins) and ion exchange chromatography (see Using ion exchange chromatography to purify a recombinantly expressed protein) are more ideally suited for a first purification step. If the goal of the experiment is to separate different oligomeric species, the initial sample should be concentrated (see TCA Precipitation or Salting out of proteins using ammonium sulfate precipitation) to a very small volume to prevent overlapping elution of two different species. If there is not a reasonable amount of separation between species, one may concentrate the sample further or use a longer column. Ideally, this experiment should be performed using an FPLC system capable of monitoring the UV absorbance of the protein(s) of interest and a fraction collector.
2. EQUIPMENT FPLC system (with UV detector and fraction collector) Gel filtration column Vacuum filtration assembly (sidearm flask and filter holder) 0.22-mm filters (for filtration assembly) Syringes 0.22-mm syringe filters
3. MATERIALS Tris-hydrochloride (Tris-HCl) Sodium chloride (NaCl) Dithiothreitol (DTT) Potassium chloride (KCl)
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Gel Filtration Kit for Molecular Weights 12,000-200,000 (Sigma cat # MWGF200, adjust if necessary for column)
3.1. Solutions & buffers Step 1 Molecular weight standards buffer Component
Final concentration
Stock
Amount
Tris–HCl, pH 7.5
50 mM
1M
200 ml
KCl
100 mM
1M
400 ml
Add water to 4 l and pass through a 0.22-mm filter
Step 2 Elution buffer Component
Final concentration
Stock
Amount
Tris–HCl, pH 7.5
50 mM
1M
50 ml
NaCl
150 mM
4M
37.5 ml
DTT
0.1 mM
1M
0.1 ml
Add water to 1 l and pass through a 0.22-mm filter
4. PROTOCOL 4.1. Preparation Obtain a reasonably pure protein sample (>90% pure) for characterization by gel filtration through another chromatography method, preferably ion exchange chromatography (see Using ion exchange chromatography to purify a recombinantly expressed protein) or affinity chromatography (see Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Immunoaffinity purification of proteins, Strep-tagged protein purification, Proteolytic affinity tag cleavage or Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins). If possible, concentrate the sample to 1 ml. If this is not possible, concentrate the sample to less than 3% of the column volume (see TCA Precipitation or Salting out of proteins using ammonium sulfate precipitation). Follow the column manufacturer’s instructions for placing the column in water. Follow the instructions of the gel filtration molecular
110
Krisna C. Duong-Ly and Sandra B. Gabelli
Figure 9.2 Flowchart of the complete protocol, including preparation.
weight kit for dissolving and filtering the standardized proteins and the blue dextran. Prepare tubes for collecting fractions.
4.2. Duration Preparation
About 1–2 days
Protocol
Varies (based on column size)
See Fig. 9.2 for the flowchart of the complete protocol.
5. STEP 1 STANDARDIZATION OF THE GEL FILTRATION COLUMN 5.1. Overview Prepare the column for the protein standards and blue dextran and determine their elution volumes in order to make a standard curve.
5.2. Duration Variable (depends on column size) 1.1 Equilibrate the column with at least 2 column volumes of the molecular weight standards buffer. The flow rate should be 0.3–0.5% of the column volume per minute. Monitor the UV absorbance and when the reading is stable, set it to ‘0.’
Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins
111
1.2 Inject 1 ml of the blue dextran solution and elute with 2 column volumes of the molecular weight standards buffer. Measure the elution volume of blue dextran to the center of the elution peak. This is the void volume. 1.3 Inject 1 ml of one of the protein standards and elute with 2 column volumes of the molecular weight standards buffer. Measure the elution volume. 1.4 Repeat Step 1.3 for the remaining protein standards. 1.5 Graph the log10 of the molecular weight of each protein standard versus the ratio of its elution volume to the void volume. Determine the line of best fit. This is the standard curve.
5.3. Tip If the UV absorbance does not stabilize after 2 column volumes, consider performing a rigorous cleaning of the column according to the manufacturer’s instructions or running more buffer over the column until the UV absorbance reading is stable.
5.4. Tip The elution volume includes the volume of sample that is injected; thus, one should set ‘0’ ml to be the time at which the injection of the sample begins.
5.5. Tip Eluting with 2 column volumes prepares the column for the next standard. Thus, a one column volume elution is sufficient for the final protein standard.
5.6. Tip This step may be skipped if the column has been standardized previously and has not suffered any changes due to repacking, etc.
5.7. Tip If a different molecular weight standards kit is used, use the buffer recommended for that kit. The buffer used in this protocol was recommended by Sigma for this particular product. See Fig. 9.3 for the flowchart of Step 1.
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Krisna C. Duong-Ly and Sandra B. Gabelli
Figure 9.3 Flowchart of Step 1.
6. STEP 2 DETERMINATION OF THE SIZES OF PROTEIN SPECIES IN A SAMPLE 6.1. Overview Purify a protein sample using gel filtration chromatography and determine the molecular weight of any species present.
6.2. Duration About 1 day 2.1 Remove the molecular weight standards buffer by washing the column with at least half a column volume of water. 2.2 Equilibrate the column with 2 column volumes of the elution buffer. When the UV absorbance reading is stable, set it to ‘0.’ 2.3 Pass the sample through a 0.22-mm syringe filter. 2.4 Inject the sample and elute with at least 1 column volume of the elution buffer. Measure the elution volume of each protein species. 2.5 Calculate the ratio of the elution volume to the void volume and use the standard curve to determine the size of the eluted species.
Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins
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6.3. Tip If the sample clogs the syringe filter, it may contain large aggregates. Use multiple syringe filters if this occurs.
6.4. Tip If the UV absorbance contains spikes or is unusually high and DTT is present, this indicates that the DTT may have oxidized. Make new buffer and add DTT right before the experiment. Avoid using buffers containing DTT that are more than 1-day old.
6.5. Tip If the column goes over pressure, decrease the flow rate. If this is a recurring problem, follow the column manufacturer’s instructions for rigorously cleaning the column.
6.6. Tip If the protein contains no Trp, it will not absorb appreciably at 280 nm. In this case, monitor the absorbance at 260 nm (for Tyr), or at another wavelength, to detect the protein.
6.7. Tip Salt (>150 mM ) is required in the protein and elution buffers to prevent protein adhesion to the beads. Additionally, if there is a difference between the conductivities
Figure 9.4 Flowchart of Step 2.
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Krisna C. Duong-Ly and Sandra B. Gabelli
of the sample and of the elution buffer, it will be apparent in the conductivity measurements at the position of the column volume. See Fig. 9.4 for the flowchart of Step 2.
REFERENCES Referenced Literature Scopes, R. (1993). Protein Purification: Principles and Practice (3rd ed.). New York: Springer. Whitaker, J. R. (1963). Analytical Chemistry, 35, 1950. Cutler, P. (1998). Size-exclusion chromatography. In R. Rapley & J. M. Walker (Eds.), Molecular Biomethods Handbook. (pp. 451–460): Humana Press.
Referenced Protocols in Methods Navigator Protein Affinity Purification using Intein/Chitin Binding Protein Tags. Purification of His-tagged proteins. Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag. Purification of GST-tagged proteins. Immunoaffinity purification of proteins. Strep-tagged protein purification. Proteolytic affinity tag cleavage. Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins. Using ion exchange chromatography to purify a recombinantly expressed protein. TCA Precipitation. Salting out of proteins using ammonium sulfate precipitation.