37
UNIT 3
CHROMATOGRAPHY OF PROTEINS
Introduction:
Even when produced by a recombinant host at high expression levels, proteins come in complex mixtures
and frequently require multiple purification steps to remove the contaminants. The protein must be
separated from hundreds of other proteins with similar properties, as well as all the lipid, nucleic acids,
and carbohydrate-containing biomolecules of the cell.
One of the most powerful methods of purification that can do this job is column chromatography. Like all
chromatography techniques, there is a stationary phase and a mobile phase in column chromatography.
The stationary phase is a fine bead particle that has defined chemical properties that proteins interact
with. The mobile phase is a buffer that the protein is soluble in. The separation of proteins on a column
relies on their different relative affinities for the stationary, compared to the mobile, phase. Proteins with
higher affinity for the stationary phase will be retained on the column while proteins with a greater affinity
for the buffer will elute from the column.
There are many types of column chromatography, based on the chemistry of the stationary phase. The
general classifications are listed below.
Table 3.1. Types of chromatography commonly used for purifying proteins
Technique
Gel Filtration (GFP), or Size Exclusion
Chromatography (SEC)
Ion exchange
Based on protein property
Size, shape
Hydrophobic interaction chromatography (HIC)
Immobilized metal affinity chromatography (IMAC)
Hydrophobicity of amino side groups on protein
surfaces
Histidine amino acids on protein surfaces
Affinity Chromatography
Biorecognition (ligand specific)
Net charge on protein surfaces
In the case of SEC, separation on the column is a function of size alone: larger molecules are excluded
from the pores of the chromatography beads and elute from the column more quickly than smaller
molecules. Here, the resolving power is a function of the length of the column that the proteins are
separated on, as well as the pore size of the beads, the flow rate of the elution buffer, and the relative
sizes of proteins being separated. Since the permeation of the beads by smaller proteins is a diffusion
dependent process, the flow rates should be slow enough to allow for this to happen. In general, the
resolution of SEC on small columns is not great, but this step can also be useful for changing the buffer
that the protein is dissolved in to the buffer used to elute the proteins from the column. Major drawbacks
to SEC is that the volume of the protein sample applied to the column must not exceed 5% of the column
volume, and the protein eluted from the column is generally diluted by a factor of 10-fold. This means
that a SEC step must usually be followed by a step that will concentrate the protein fraction.
The other forms of chromatography listed in Table 3.1 are a form of “adsorption” chromatography, in that
the protein that is applied to the column matrix will be adsorbed to the matrix, allowing separation from
proteins that have no affinity to the column. The proteins left on the column can then be eluted by
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
38
changing the buffering conditions of the mobile phase. A gradual change in buffering conditions
can elute proteins one at a time, allowing their separation from each other.
Adsorption chromatography can be used to concentrate proteins, since a large volume of protein can be
applied to the column without affecting the amount of protein adsorbed to the column matrix. Careful
selection of elution conditions can remove the protein from the column in a much smaller volume. Since
adsorption chromatography allow for both the isolation and concentration of a target protein, it is
sometimes referred to a “capture” phase of protein purification.
There are different types of materials that chromatography beads are made from. The matrix from which
the adsorbent is made has important qualities. If must be readily porous for large proteins, yet sufficiently
rigid to sustain hydrostatic pressure of elution buffers. At the same time, the bead material itself must not
have a high affinity for proteins: the adsorption of proteins must rely solely on the ligand coating the
bead, whether that be an ion exchange group, a hydrophobic group, or an affinity label. The most
common chromatographic materials used to make beads are insoluble polysaccharides such as cellulose,
dextrans, or agarose. Sephadex is a bead-formed gel prepared by cross-linking dextran with
epichlorohydrin. The dextran hydroxyl group renders the gel extremely hydrophilic, and the degree of
cross-linking determines the pore size of the gel. The lower the cross-linking, the more porous the bead
is and the larger the proteins that have access to its surfaces, both interior and exterior. Unfortunately,
the more porous bead have less mechanical strength and are compressed by high hydrostatic pressures
found in large columns or when elution buffers are pumped too quickly. Cross-lined agarose-type beads,
such as Sepharose, have greater mechanical stability and are preferred for larger columns of for faster
elution rates. Both types of beads are biodegradable, so must be stored with a biocide such as sodium
azide or high concentrations of alcohol. Although cold storage is best, the beads are damaged by
freezing.
There is no single best way to purify all proteins. Finding an optimal protein purification strategy requires
trial and error, because the best method for purification depends on the properties of protein being
isolated as well as the proteins and other contaminants that the protein must be isolated from. Different
chromatography methods are tested and compared for their suitability in purifying each new protein. In
choosing the best types of chromatography to use, there are at least five factors that must be evaluated:
 resolution of the method
 capacity of the method
 speed of the method
 % recovery of protein
 cost
In this lab exercise, we will compare HIC with an affinity chromatography technique, immobilized metal
affinity chromatography (IMAC) in the purification of the GFP that you isolated in Lab Exercise 2.
Hydrophobic interaction chromatography (HIC)
Separation by HIC is based on the reversible interaction between a protein and the hydrophobic surface
of a chromatographic medium. This interaction is enhanced at high ionic strength buffer solution (e.g. 1.5
M ammonium sulphate). Proteins bind to the column as they are loaded, and proteins with low affinity
and other contaminants are washed from the column with high ionic strength buffer. Conditions are then
altered so that the bound substances are eluted differentially, usually by decreasing the salt concentration
of the elution buffer. Changes are made stepwise or with a continuously decreasing salt gradient. Target
proteins are concentrated during binding and collected in a purified, concentrated form. Some proteins
with extremely high affinity to the HIC matrix must be eluted with buffers of reduced polarity (e.g. an
ethylene glycol gradient up to 50%). Some will elute by adding chaotropic species (urea, guanidine
hydrochloride) or detergents. Sometimes a change of pH or temperature can be used to affect the elution
of proteins, as well. In general, hydrophobic interactions increase in strength with increasing
temperature; there can be a 20-30% reduction in binding when the temperature is changed from 20 to
4oC. Generally, the strength of the interaction between proteins and hydrophobic ligands decreases with
increasing pH. This is presumably because of an increase in the hydrophilicity of proteins due to the
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
39
titration of charged groups. The effect of pH is different for different proteins, and thus elution profiles
may be improved by changing the elution pH. However, the effect of pH on elution from HIC is not that
great, and it is best to work with the pH range that the protein is stable at.
The most popular HIC resins are cross-linked agarose gels to which hydrophobic ligands have been
covalently attached. The choice of ligand will determine the degree of hydrophobicity of the HIC resin.
The most popular ligands include, in the order of increasing hydrophobicity:
methyl- < phenyl- < octyl-.
If a protein binds too tightly, requiring a harsh elution buffer, a less hydrophobic matrix might work better.
A variety of salts may be used for loading and elution. In general the effects of various salts on HIC
mimic their effects on salting out proteins. Those salts which are most effect in salting out are most
effective at binding proteins to hydrophobic matrices. Thus, ammonium sulfate is generally a good salt
for binding most proteins to an HIC column.
Immobilized Metal Affinity Chromatography (IMAC)
IMAC stationary phases are designed to chelate metal ions that have selectivity for specific groups on
protein surfaces. A strong chelating group is added to the chromatographic matrix, such that when the
metal ions are introduced, they are complexed leaving part of the coordination sphere of the metal ion
free. Ligands will therefore coordinate with these metals, including residues in proteins. Histidine, and to
a lesser extent cysteine and tryptophan, residues on the protein’s surface are mainly responsible for the
interaction with IMAC residues. Most proteins have these amino acid residues, and in some cases a
single histidine in a small protein is sufficient to cause an interaction strong enough to bind the protein to
an IMAC column.
The nature of the metal and the way it is coordinated on the column also influences the strength of
interactions, so that a whole range of selectivity is introduced. Most work has used divalent metals in the
first transition series through zinc. Although much of the earlier work concentrated on the use of Cu +2 and
Zn+2 complexes, Co+2, Ni2+, and even Fe3+ complexes are commonly used today.
Operation of an IMAC column involves first “charging up” the chelating adsorbent with metal; a small
pulse of quite concentrated (50 mM) metal salt is passed through, followed by water. The column
acquires a color as themetal ions attach (except for zinc). A prewash with intended elution buffers is
usually carried out. Sample buffers contain salt (up to 1 M) to minimize nonspecific ion-exchange effects,
and adsorption of proteins is maximal at higher pH. Elution is normally either by lowering of pH to
protonate the donor histidine groups on the adsorbed protein, or by use of a stronger complexing agent
such as imidazole. Because some eluting buffers also cause some leakage of metal ions, it can be a
good idea to have some adsorbent that has not been charged with the metal on the bottom of the column
to trap this leakage.
Safety Considerations:
Wear closed toed shoes whenever you are in lab.
Wear gloves while handling the acid to adjust the pH.
Wear gloves when handling hazardous chemicals and dispose in hazardous waste bin.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
40
MEDIA PREP FORM
Control #
Name of Solution/Media:
Amount prepared:
Date:
Preparers(s):
Component
Brand/Item #
Storage
conditions/
date received
FW or initial
concentration
Balance used
Calibration status
pH meter used
Calibration status
Initial pH
Final pH
Adjusted pH with
Prep temperature
Sterilization procedure
Storage conditions
Amount
used
Final
concentration
Calculations/Comments:
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
41
MEDIA PREP FORM
Control #
Name of Solution/Media:
Amount prepared:
Preparers(s)
Date:
:
Component
Brand/Item #
Storage
conditions/
date received
FW or initial
concentration
Balance used
Calibration status
pH meter used
Calibration status
Initial pH
Final pH
Adjusted pH with
Prep temperature
Sterilization procedure
Storage conditions
Amount
used
Final
concentration
Calculations/Comments:
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
42
Protocol:
FIRST DAY
On the first day, you will need to set up your solutions and columns for protein purification. There are 3
different column purifications that will be compared; each group will perform one purification step, and
comparisons will be made between the different groups in the classroom. All purifications must be
included in the lab report in order to make comparisons, so information must be exchanged between
groups to be included in individuals’ reports. Students may collaborate in the preparation of the following
solutions for use by the entire class. Part of this collaboration is the sharing of the Media Prep Form
describing the preparation of each solution in for individual lab reports. Students using a solution should
record its control number in their lab report.
1. The following reagents will be used for purification of GFP by chromatography. Groups may share
responsibility for making the different reagents, but everyone must have a copy of the prep form for
each solution that is used during the experiment. Make sure you double check your calculations with
your partners. When finished, dispense the solution into bottles and label each with the reagent,
your initials and the date. Make out a Media Prep form. Make up the stock solutions first, and make
the working solutions from the stocks when necessary.
Solution
Final concentration
Comments
4.0 M
Final
volume
100 mL
Ammonium sulfate
stock solution
Polyethylene glycol
(PEG)
5% (w/v)
20 mL
Store at room temperature.
Sodium phosphate in
purified H2O, pH 7.0
1.0 M
100 mL
Store refrigerated.
Prepared in Lab 2A
1M NaCl stock solution
(50 mL)
1.0 M
50 mL
Store at room temperature.
IMAC equilibration
buffer
20 mM Na phosphate
0.5 M NaCl
pH 7.0
100 mL
From stocks. Store refrigerated.
IMAC elution buffer #1
0.1 M Na acetate
0.5 M NaCl
pH 4.0
20 mL
Start with 0.1M acetic acid, adjusting pH
with NaOH. Add NaCl from stock. Store
refrigerated.
IMAC elution buffer #2
20 mM Na phosphate
0.4 M imidazole
pH 7.0
20 mL
HIC equilibration buffer
20 mM Na phosphate
2 M ammonium sulfate
pH 7.0
100 mL
Dilute phosphate buffer from stock. Add
the imidazole and adjust the pH before
adjusting the final volume. Store
refrigerated.
From stocks. Store refrigerated.
HIC elution buffer #1
20 mM Na phosphate,
1 M ammonium sulfate
pH 7.0
20 mM Na phosphate,
1 % PEG
pH 7.0
20 mL
From stocks. Store refrigerated.
20 mL
From stocks. Store refrigerated.
HIC elution buffer #1
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
Store at room temperature
ACC Lab Manual, 2nd Edition
2007
43
2. Set up your columns. These resins are high-capacity, and you should not need more than 2-3 mL
of column bed volume. Select your column size accordingly. Fix your column to a ring stand so that
the column is vertical. Plumb your column with the narrowest tubing that you can fit to it. If possible,
find a stop cock for the bottom fitting so that you can turn off your column when necessary. If you do
not have a stock cock for your column, you will need clamps to stop the flow of buffer through your
column. You will need to place your buffers above your column to feed through the column by gravity
flow.
SECOND DAY
You will compare two types of chromatography with small testing columns, one with HIC phenyl
Sepharose resin (phenyl agarose may be substituted) and two with a nickel IMAC resins (with two
different types of elution strategies). After packing columns with resins in the appropriate equilibration
buffer, you will apply your cell extract to the column to adsorb the proteins. After nonadsorbed proteins
and other contaminants are washed from the column with equilibration buffer, proteins will be eluted by
gradually changing the elution buffer. You will be able to immediately tell where the GFP is at any point
by simply looking for fluorescence under a black light. Fractions containing GFP will be saved for later
analysis by gel electrophoresis.
Part I. HIC: Phenyl Sepharose Chromatography
1. Phenyl Sepharose is stored in a sodium azide solution to prevent microbial growth, and the sodium
azide must be removed prior to its use. Dispense the appropriate amount of resin by repeatedly
inverting the phenyl Sepharose container to resuspend the beads and immediately pipetting or
pouring off as much as you will need for this lab (about 5-10 mL per column). Transfer into a 15 mL
conical centrifuge tube. Measurements do not have to be exact.
2. Allow the suspension to resettle. This should take 5 minutes or less. You may centrifuge the mixture
for a minute in a clinical centrifuge to settle the beads faster.
3. Remove the sodium azide used to preserve the phenyl Sepharose by pipetting it off the top. Be
careful to draw up only liquid.
4. Add 5 mL of HIC equilibration buffer (2M ammonium sulfate in 20 mM Na phosphate pH 7.0) and mix
well by gentle inversion. This removes most, but not all, of the sodium azide. The rest will be
removed in subsequent steps.
5. Allow the beads to settle or centrifuge to settle the beads.
6. Remove the buffer from the top and add back fresh equilibration buffer. This should remove all of the
sodium azide that was used as a preservative for the resin.
7. Prepare a column for assembly by clamping a BioRad Poly-Prep column to a ring stand. (If
devising your column from a pasteur pipette, use the tip of a straightened large paper clip to push a
small plug of glass wool into the tip of a pasteur pipette or dropper to form a “frit” for holding resin in
the column. Do not push the glass wool too tight or it will slow the flow of buffer through the column.
Also, if you have no ring stand and clamps, you can hold the column in a vertical position by using a
styrofoam cup with the sides cut out as a stand. If you have a ring stand but not clamps, use twist
ties to affix the column to a ring stand.)
8. Place a 50 mL beaker or plastic cup under the column to collect waste liquids. Keep collecting liquids
in this container until the instructions call for collecting column fractions.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
44
9. Use a transfer pipette or pasteur pipette to add approximately 1 mL of equilibration buffer to the
column to soak the frit (or glass wool plug) first in order to remove any air bubbles. A standard black
dropper bulb draws up about 1 mL. (If the liquid doesn’t start to drop out of the pipette you have
pushed the glass wool plug in too tightly. Start again.)
10. As the equilibration buffer is flowing through the frit and before the column goes dry, load your phenyl
Sepharose resin. Gently invert the phenyl Sepharose slurry that you have prepared in order to
resuspend the beads. With the tip of the pasteur pipette or dropper touching the side of the column,
slowly add about 8 mL of slurry. Avoid introducing any air bubbles into the column. The excess
liquid will drain through the column and into the waste container beaker below.
11. Let the equilibration buffer flow through the column to equilibrate the column conditions and to pack
the column with beads. DO NOT ALLOW THE COLUMN TO GO DRY AT ANY TIME! Make sure the
bed height of the column is about 4-5 cm and the top of the column bed is level. The exact height is
not important, but make sure that your beads fill the lower, more constricted end of the column. You
need enough resin in the column to bind all of the protein molecules you will be loading. If there are
more protein molecules than resin beads available to bind them the extra proteins will pass right
through the column without being separated. If the column is too short after packing, add more slurry.
12. The general rule in equilibrating columns is to add 2 column volumes of equilibration buffer and let it
wash through. Gently load 10 mL of equilibration buffer onto the column without introducing air to the
buffer and without disturbing the column bed. You can do this by placing the tip of the pipette or
dropper on the wall of the column near to top of the column bed and allowing the buffer to flow gently
down the inside of the column. If there is not enough room to add all of the buffer at once, keep
adding more as it drains through the column. Allow the buffer to drain through the matrix into the
beaker or cup.
13. While your column is equilibrating, prepare your cell extract for loading. It must be thawed and
adjusted to 2M in ammonium sulfate. Transfer 0.5 mL of the cell extract to a fresh microcentrifuge
tube and add an equal volume of 4 M ammonium sulfate. Any precipitated matter that may have
formed from the freeze-thaw procedure or the addition of ammonium sulfate must be removed by
centrifugation (full speed 10 minutes in refrigerated micrcentrifuge). If there is no visible precipitation,
then there is no need to centrifuge the samples. Carefully transfer the sample supernatant or filtrate
to a fresh microcentrifuge tube, using a pasteur pipet. Keep your cell extract cold by placing it on
crushed ice.
14. When the last of the equilibration buffer has entered the column, slowly load 1.0 mL of your cell
extract onto the top of column. Be careful not to disturb the chromatography bed or to introduce air
bubbles.
15. When the cell extract has entered the column bed, carefully add 5 mL of equilibration buffer to the top
and collect the column effluent into a test tube marked “wash”, You should see the GFP getting
“trapped” in the first layers of the column by observing the column under a black light. This is
because the high salt concentration in the equilibration buffer increases the hydrophobic interactions
between the proteins in the sample with the phenyl groups of the resin. They are attracted to the
hydrophobic beads in the column resin and bind to them.
16. When the wash buffer has eluted from the column, add the 5 mL of 1.0 M ammonium sulfate elution
buffer (HIC Elution Buffer #1) that you have prepared to the column. Be careful not to disturb the
chromatography resin and be careful not to allow the column to go dry. At this point, you do not know
where the GFP will elute, so you need to start collecting fractions for later analysis. Collect 1.5 mL
fractions into microcentrifuge tubes. Label your collection fractions by number and store them on
crushed ice.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
45
17. When the HIC Elution Buffer #1 has eluted from the column, repeat the elution process using the 5
mL of the HIC Elution Buffers #2. Continue to collect 1.5 mL column fractions in microcentrifuge
tubes.
18. View your collected column fraction under UV light to locate the fraction(s) containing GFP. Label the
fraction with its contents, the date, and your group, and freeze for later analysis.
Part II. IMAC: Nickel Immobilized Affinity Chromatography with Imidazole
Gradient Elution
1. The nickel immobilized chromatography resin is stored in an alcohol solution to prevent microbial
growth, and the alcohol must be removed prior to its use. Dispense the appropriate amount of resin
by repeatedly inverting the container to resuspend the beads and immediately pipetting or pouring off
as much as you will need for this lab (about 10 mL per column). Transfer into a 15 mL conical
centrifuge tube. Measurements do not have to be exact.
2. Allow the suspension to resettle. This should take 5 minutes or less. You may centrifuge the mixture
for a minute in a clinical centrifuge to settle the beads faster.
3. Remove the ethanol used to preserve the chromatography beads by pipetting it off the top. Be
careful to draw up only liquid.
4. Add 5 mL of IMAC equilibration buffer (20 mM Na phosphate, 0.5 M NaCl pH 7.0) and mix well by
gentle inversion. This removes most, but not all, of the ethanol. The rest will be removed in
subsequent steps.
5. Allow the beads to settle or centrifuge to settle the beads.
6. Remove the buffer from the top and add back fresh equilibration buffer. This should remove all of the
ethanol that was used as a preservative for the resin.
7. Prepare a column for assembly by clamping a BioRad Poly-Prep column to a ring stand. (If
devising your column from a pasteur pipette, use the tip of a straightened large paper clip to push a
small plug of glass wool into the tip of a pasteur pipette or dropper to form a “frit” for holding resin in
the column. Do not push the glass wool too tight or it will slow the flow of buffer through the column.
Also, if you have no ring stand and clamps, you can hold the column in a vertical position by using a
styrofoam cup with the sides cut out as a stand. If you have a ring stand but not clamps, use twist
ties to affix the column to a ring stand.)
8. Place a 50 mL beaker or plastic cup under the column to collect waste liquids. Keep collecting liquids
in this container until the instructions call for collecting column fractions.
9. Use a transfer pipette or pasteur pipette to add approximately 1 mL of equilibration buffer to the
column to soak the frit (or glass wool plug) first in order to remove any air bubbles. A standard black
dropper bulb draws up about 1 mL. (If the liquid doesn’t start to drop out of the pipette you have
pushed the glass wool plug in too tightly. Start again.)
10. As the equilibration buffer is flowing through the frit and before the column goes dry, load your nickel
immobilized chromatography resin. Gently invert the slurry that you have prepared in order to
resuspend the beads. With the tip of the pasteur pipette or dropper touching the side of the column,
slowly add about 8 mL of slurry. Avoid introducing any air bubbles into the column. The excess
liquid will drain through the column and into the waste container beaker below.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
46
11. Let the equilibration buffer flow through the column to equilibrate the column conditions and to pack
the column with beads. DO NOT ALLOW THE COLUMN TO GO DRY AT ANY TIME! Make sure
the bed height of the column is about 4-5 cm and the top of the column bed is level. The exact height
is not important, but make sure that your beads fill the lower, more constricted end of the column. You
need enough resin in the column to bind all of the protein molecules you will be loading. If there are
more protein molecules than resin beads available to bind them the extra proteins will pass right
through the column without being separated. If the column is too short after packing, add more slurry.
12. The general rule in equilibrating columns is to add 2 column volumes of equilibration buffer and let it
wash through. Gently load 10 mL of equilibration buffer onto the column without introducing air to the
buffer and without disturbing the column bed. You can do this by placing the tip of the pipette or
dropper on the wall of the column near to top of the column bed and allowing the buffer to flow gently
down the inside of the column. If there is not enough room to add all of the buffer at once, keep
adding more as it drains through the column. Allow the buffer to drain through the matrix into the
beaker or cup.
13. While your column is equilibrating, prepare your cell extract for loading. It must be thawed and any
precipitated matter that may have formed from the freeze-thaw treatment must be removed by
centrifugation (full speed 10 minutes in refrigerated micrcentrifuge). If there is no visible precipitation,
then there is no need to centrifuge the samples. Carefully transfer the sample supernatant or filtrate
to a fresh microcentrifuge tube, using a pasteur pipet. Keep your cell extract cold by placing it on
crushed ice.
14. When the last of the equilibration buffer has entered the column, slowly load 0.5 mL of your cell
extract onto the top of column. Be careful not to disturb the chromatography bed or to introduce air
bubbles.
15. When the cell extract has entered the column bed, carefully add 5 mL of equilibration buffer to the
top slowly so that you do not disturb the column bed. Collect the column effluent into a test tube
marked “wash”. You should see the GFP getting “trapped” in the first layers of the column by
observing the column under a black light. Watch the column so that you do no allow the column to go
dry.
16. While you are washing impurities from the column with equilibration buffer, prepare a couple of
solutions (5 mL each) of increasing imidazole concentration for eluting adsorbed proteins from the
column by a step gradient. You can make the following solutions by diluting IMAC equilibration buffer
with the IMAC elution buffer #2 (0.4 M imidazole in 20 mM Na phosphate pH 7.0) to make to following
concentrations of imidazole:
 0.15 M imidazole
 0.3 M imidazole
17. When the wash buffer has eluted from the column, carefully add the 5 mL of the 0.15 M imidazole
elution buffer that you have prepared to the top of the column. Be careful not to disturb the
chromatography resin and be careful not to allow the column to go dry. At this point, you do not know
where the GFP will elute, so you need to start collecting fractions for later analysis. Collect 1.5 mL
fractions into microcentrifuge tubes. Label your collection fractions by number and store them on
crushed ice.
18. When the 0.15 M imidazole elution buffer has eluted from the column, repeat the elution process
using the 5 mL of 0.3 M imidazole elution buffer. Be careful not to disturb the column bed and to not
allow the column to go dry. Continue to collect 1.5 mL column fractions in microcentrifuge tubes.
19. View your collected column fraction under UV light to locate the fraction(s) containing GFP. Label the
fraction with its contents, the date, and your group, and freeze for later analysis.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
47
Part III. IMAC: Nickel Immobilized Affinity Chromatography with pH Gradient
Elution
1. The nickel immobilized chromatography resin is stored in an alcohol solution to prevent microbial
growth, and the alcohol must be removed prior to its use. Dispense the appropriate amount of resin
by repeatedly inverting the container to resuspend the beads and immediately pipetting or pouring off
as much as you will need for this lab (about 10 mL per column). Transfer into a 15 mL conical
centrifuge tube. Measurements do not have to be exact.
2. Allow the suspension to resettle. This should take 5 minutes or less. You may centrifuge the mixture
for a minute in a clinical centrifuge to settle the beads faster.
3. Remove the ethanol used to preserve the chromatography beads by pipetting it off the top. Be
careful to draw up only liquid.
4. Add 5 mL of IMAC equilibration buffer (20 mM Na phosphate, 0.5 M NaCl pH 7.0) and mix well by
gentle inversion. This removes most, but not all, of the ethanol. The rest will be removed in
subsequent steps.
5. Allow the beads to settle or centrifuge to settle the beads.
6. Remove the buffer from the top and add back fresh equilibration buffer. This should remove all of the
ethanol that was used as a preservative for the resin.
7. Prepare a column for assembly by clamping a BioRad Poly-Prep column to a ring stand. (If
devising your column from a pasteur pipette, use the tip of a straightened large paper clip to push a
small plug of glass wool into the tip of a pasteur pipette or dropper to form a “frit” for holding resin in
the column. Do not push the glass wool too tight or it will slow the flow of buffer through the column.
Also, if you have no ring stand and clamps, you can hold the column in a vertical position by using a
styrofoam cup with the sides cut out as a stand. If you have a ring stand but not clamps, use twist
ties to affix the column to a ring stand.)
8. Place a 50 mL beaker or plastic cup under the column to collect waste liquids. Keep collecting liquids
in this container until the instructions call for collecting column fractions.
9. Use a transfer pipette or pasteur pipette to add approximately 1 mL of equilibration buffer to the
column to soak the frit (or glass wool plug) first in order to remove any air bubbles. A standard black
dropper bulb draws up about 1 mL. (If the liquid doesn’t start to drop out of the pipette you have
pushed the glass wool plug in too tightly. Start again.)
10. As the equilibration buffer is flowing through the frit and before the column goes dry, load your nickel
immobilized chromatography resin. Gently invert the slurry that you have prepared in order to
resuspend the beads. With the tip of the pasteur pipette or dropper touching the side of the column,
slowly add about 8 mL of slurry. Avoid introducing any air bubbles into the column. The excess
liquid will drain through the column and into the waste container beaker below.
11. Let the equilibration buffer flow through the column to equilibrate the column conditions and to pack
the column with beads. DO NOT ALLOW THE COLUMN TO GO DRY AT ANY TIME! Make sure
the bed height of the column is about 4-5 cm and the top of the column bed is level. The exact height
is not important, but make sure that your beads fill the lower, more constricted end of the column. You
need enough resin in the column to bind all of the protein molecules you will be loading. If there are
more protein molecules than resin beads available to bind them the extra proteins will pass right
through the column without being separated. If the column is too short after packing, add more slurry.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
48
12. The general rule in equilibating columns is to add 2 column volumes of equilibration buffer and let it
wash through. Gently load 10 mL of equilibration buffer onto the column without introducing air to the
buffer and without disturbing the column bed. You can do this by placing the tip of the pipette or
dropper on the wall of the column near to top of the column bed and allowing the buffer to flow gently
down the inside of the column. If there is not enough room to add all of the buffer at once, keep
adding more as it drains through the column. Allow the buffer to drain through the matrix into the
beaker or cup.
13. While your column is equilibrating, prepare your cell extract for loading. It must be thawed and any
precipitated matter that may have formed from the freeze-thaw treatment must be removed by
centrifugation (full speed 10 minutes in refrigerated micrcentrifuge). If there is no visible precipitation,
then there is no need to centrifuge the samples. Carefully transfer the sample supernatant or filtrate
to a fresh microcentrifuge tube, using a pasteur pipet. Keep your cell extract cold by placing it on
crushed ice.
14. When the last of the equilibration buffer has entered the column, slowly load 0.5 mL of your cell
extract onto the top of column. Be careful not to disturb the chromatography bed or to introduce air
bubbles.
15. When the cell extract has entered the column bed, carefully add 5 mL of equilibration buffer to the
top, slowly so that you do not disturb the column bed. Collect the column effluent into a test tube
marked “wash”. You should see the GFP getting “trapped” in the first layers of the column by
observing the column under a black light. Watch the column to make sure that it does not go dry.
16. While you are washing impurities from the column with equilibration buffer, prepare a couple of
solutions (5 mL each) of decreasing pH for eluting adsorbed proteins from the column by a step
gradient. You can make the following solutions by diluting IMAC equilibration buffer with the IMAC
elution buffer #1 (0.1 M Na acetate, 0.5 M NaCl pH 4.0) in the following ratios:
Elution gradient
step
First
IMAC equilibration buffer
IMAC elution buffer #1
2.5 mL
2.5 mL
Second
1 mL
4 mL
17. When the wash buffer has eluted from the column, add the 5 mL of the first elution gradient step
buffer that you have prepared to the column. Be careful not to disturb the chromatography resin and
be careful not to allow the column to go dry. At this point, you do not know where the GFP will elute,
so you need to start collecting fractions for later analysis. Collect 1.5 mL fractions into
microcentrifuge tubes. Label your collection fractions by number and store them on crushed ice.
18. When the first elution gradient buffer has eluted from the column, repeat the elution process using the
5 mL of second elution gradient buffer. Continue to collect 1.5 mL column fractions in microcentrifuge
tubes.
19. When the second elution gradient buffer has eluted from the column, repeat the elution process using
5 mL of IMAC elution buffer #1. Continue to collect 1.5 mL column fractions in microcentrifuge tubes.
20. View your collected column fraction under UV light to locate the fraction(s) containing GFP. Label the
fraction with its contents, the date, and your group, and freeze for later analysis.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
49
ALTERNATIVE ANALYSIS (if time):
A more quantitative approach to analyzing which chromatography fractions contain GFP would be
to determine fluorescence in a fluorimeter. This can be used to determine the following results:
a. How efficient your elution conditions were at releasing GFP from the column in a narrow
fraction, producing a small volume of highly concentrated GFP.
b. The quantitative yield of GFP from the column. This can be determined by the
fluorescence multiplied by the total volume of GFP collected from the column. The
percent yield can be done by comparing the fluorescence of the sample loaded multiplied
by the volume of sample loaded onto the column.
CLEAN UP
Chromatographic matrices are very expensive and with proper care, can be reused for an
indefinite period of time. In order to properly care for the column matrix, you should “clean up”
any tightly adsorbed proteins from the column with an extreme-pH buffer, when possible. Many
matrices cannot withstand such a harsh treatment, so make sure that you do not use this
approach unless the product literature condones it. It is very important to thoroughly remove this
highly acidic or basic buffer from the column before storage.
Since most chromatographic matrices used for protein purifications are made from highly
biodegradable materials such as dextrans, it is very important that you store these materials in a
bacteriostatic buffer. Microbial growth can be effectively inhibited with a high concentration of
ethanol or a dilute solution of sodium azide. Be sure to check the product literature for your
chromatographic matrix to decide the best way to preserve your material.
1. Phenyl Sepharose columns: run 5 mL of 10mM NaOH through the column, followed by 20 mL
of deionized water. Allow the column to go dry and dispense the beads into a plastic bottle by
inverting it and rinsing it with a wash bottle or syringe of deionized water. Adjust the volume
with ethanol to make it 20% ethanol. Label the bottle as used, washed phenyl Sepharose.
Include your names and date on the label, and place in the refrigerator for long-term storage.
2. IMAC columns: run 20 mL of deionized water through the column. Allow the column to go dry
and dispense the beads into a plastic bottle by inverting it and rinsing it with a wash bottle or
syringe of deionized water. Adjust the volume with ethanol to make it 20% ethanol. Label the
bottle as used, washed nickel-IMAC. Include your names and date on the label, and place in
the refrigerator for long-term storage.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
50
Questions for Unit 3
1. Read Chapter 27 “Introduction to Bioseparations” by Seidman & Moore in Basic Laboratory Mehods
for Biotechnology, section II. Chormatography on pp 584-588. Answer the following questions.
a. Chromatography is an expensive purification step. Explain why it is commonly used in the
purification of proteins.
b. What are the mobile and stationary phases of the chromatography columns that were used in this
lab exercise?
c. What is the basis of protein separations in the chromatography columns that were used in this lab
exercise?
d. What is HPLC, and what are some advantages of HPLC chromatography, compared to the
columns that you ran in this exercise?
e. What gives rise to the higher resolution of purifications in HPLC?
f. Why do HPLC columns run at such high pressures?
g. What are some reasons that a protein purification strategy should be designed to have the fewest
possible number of purification steps?
h. What measures should be taken to stabilize proteins during purification procedures?
i. What are some special considerations that must be considered when scaling up a protein
purification procedure into a production facility?
2. Compare the results from the three column purifications that were investigated by filling out the
following table.
Column purification
Fraction(s) where GFP
eluted
Volume of GFP
fraction
Relative fluorescence
of GFP fraction
HIC-phenyl Sepharose
IMAC-imidazole elution
IMAC-pH gradient elution
3. Which procedure had the best results? Explain your reasoning, discussing the relative GFP
concentrations and the relative volumes that the GFP eluted in. Remember that the goals of
chromatography include a high overall yield as well as a concentration of the molecule that you wish
to purify.
4. What might you do to improve the purification of GFP by HIC and IMAC, based on the class results?
5. Go the National Center for Biological Information (NCBI) at www.ncbi.nlm.nih.gov and search for the
amino acid sequence of the green fluorescent protein.
a. How many amino acids are there in the GFP?
b. What amino acids, in the GFP, are hydrophobic?
c. What percentage of the GFP amino acids is hydrophobic?
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007
51
References
Chalfie, Martin and Steven Kain, Green Fluorescent Protein, Properties, Applications and Protocols
Wiley-Liss, 1998.
Doonan, S (ed.) Protein Purification Protocols in Methods in Molecular Biology (Vol. 59) Humana Press
(1996)
Price, N,C, (Ed.) Proteins Labfax. Academic Press. (1996)
Scopes, R.K. Protein Purification: Principles and Practice (2 nd ed) Springer-Verlag (1987)
Sommer, C.A., F.H. Silva, and M.T.M. Novo. Teaching Molecular Biology to Undergraduate Biology
Students. Bichem & Mol Biol Ed. 32(1):7-10 (2004)
Walmsley, R.M., N. Billinton, and W.-D. Heyer. Yeast functional analysis report: green fluorescent
protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae. Yeast
13:1535-1545 (1997)
Walsh, Gary. Biopharmaceuticals: Biochemistry & Biotechnology. (2nd ed). John Wiley & Sons (2003)
Walsh, Gary, and Denis Headon. Protein Biotechnology. John Wiley & Sons. 1994
BITC2411 Biotechnology Laboratory Instrumentation
Unit 3. Chromatography of Proteins
ACC Lab Manual, 2nd Edition
2007