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: gabelli@jhmi.edu

Contents
1. Theory 106
2. Equipment 108
3. Materials 108
3.1 Solutions & buffers 109
4. Protocol 109
4.1 Preparation 109
4.2 Duration 110
5. Step 1 Standardization of the Gel Filtration Column 110
5.1 Overview 110
5.2 Duration 110
5.3 Tip 111
5.4 Tip 111
5.5 Tip 111
5.6 Tip 111
5.7 Tip 111
6. Step 2 Determination of the Sizes of Protein Species in a Sample 112
6.1 Overview 112
6.2 Duration 112
6.3 Tip 113
6.4 Tip 113
6.5 Tip 113
6.6 Tip 113
6.7 Tip 113
References 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 # 2014 Elsevier Inc. 105

ISSN 0076-6879 All rights reserved.
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106 Krisna C. Duong-Ly and Sandra B. Gabelli

using this column. Modifications may be made to the buffer to accommodate the pro-
tein of interest and the separation range of the column.

1. THEORY
Gel filtration chromatography, also known as size exclusion chroma-
tography, is used to separate molecules of different sizes. In addition to sep-
arating 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 vol-
ume, 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 fig-
ure 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 glob-
ular. 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 dex-
tran (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 resolu-
tion 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
108 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 olig-
omeric species, the initial sample should be concentrated (see TCA Precip-
itation 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)
Gel Filtration Chromatography (Size Exclusion Chromatography) of Proteins 109

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 Recombi-
nant Proteins). If possible, concentrate the sample to 1 ml. If this is not pos-
sible, concentrate the sample to less than 3% of the column volume (see
TCA Precipitation or Salting out of proteins using ammonium sulfate pre-
cipitation). 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 deter-
mine 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 col-
umn 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 vol-
umes 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 vol-
umes 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.
112 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 113

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.

114 Krisna C. Duong-Ly and Sandra B. Gabelli

of the sample and of the elution buffer, it will be apparent in the conductivity mea-
surements 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.