Downloaded from http://cshprotocols.cshlp.

org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

Protocol

Purification of Polyhistidine-Tagged Proteins by Immobilized

Metal Affinity Chromatography
Clara L. Kielkopf, William Bauer, and Ina L. Urbatsch

Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for
divalent metal cations (usually Ni2+) immobilized as transition metal chelate complexes on a chro-
matography resin. The main protocol here is optimized for use of Ni2+–NTA resin to purify soluble
6xHis-tagged proteins by a straightforward batch method during the binding step, followed by gravity
flow for washes and elution. This protocol does not require any specialized equipment other than a
simple glass or plastic column. IMAC resins can be used in multiple formats, including batch, gravity
flow, centrifuge columns, and fast performance liquid chromatography (FPLC) systems. FPLC systems
are designed specifically for the chromatographic separations of proteins and other biomolecules.
These systems typically contain multiple pumps, an in-line UV absorption monitor, conductivity
meter, pH meter, fraction collector, and other options that allow for the simultaneous purification,
analysis, and fractionation of the sample. When linked with the appropriate instruments, an FPLC
can become a high-precision, automated instrument that separates proteins at a high resolution. An
alternative protocol is included here that describes 6xHis-tagged protein purification using FPLC.
Procedures for the cleaning and regeneration of the IMAC resin for reuse are also described, and,
finally, considerations for storing purified proteins are discussed.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
Appropriate chromatographic buffer (for resin regeneration in Steps 17–19 only)
β-mercaptoethanol (5 mM) (optional)
Low concentrations (1–5 mM) of β-mercaptoethanol may be included in the lysate, binding, wash, elution, and
dialysis buffers when cysteines are present within the target protein, without reducing the metal on the resin.

Cell lysate
Coomassie Brilliant Blue
Dialysis buffer and Wash buffer 2 <R>
When a second round of IMAC is not planned, it is advisable to include 0.5 mM EDTA (pH 8.0) to facilitate metal
ion removal. Protease inhibitors and β-mercaptoethanol also may be added depending on the protease suscept-
ibility and cysteine content of the protein.

From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
© 2020 Cold Spring Harbor Laboratory Press
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194

205
 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

C.L. Kielkopf et al.

Ethanol (20%, 25%, 50%, 75%, and 100%; v/v) (for resin regeneration in Steps 17–19 only)
IMAC binding buffer for protein purification <R>
The binding buffer contains 10 mM imidazole to minimize binding of untagged, contaminating proteins; increase
the capacity of the resin for the tagged protein; and increase purity with fewer steps. If the tagged protein does not
bind under these conditions, the amount of imidazole should be reduced (1–5 mM). Conversely, if the target
protein elutes at significantly higher imidazole concentrations, it may be possible to reduce contaminants
by further including up to 30 mM imidazole in the IMAC Binding or Wash buffers. The same concentration of
imidazole should be present in the clarified cell lysate.
A 1 M stock solution of imidazole with the pH adjusted to 8.0 using HCl can be conveniently diluted to the desired
concentrations in the Binding, Wash, and Elution buffers. A high-purity grade (>99% purity) of imidazole is
recommended (e.g., Sigma-Aldrich/Fluka BioUltra).

IMAC imidazole elution buffer<R>

Elution can be accomplished with either a step gradient or can be optimized by testing a series of different
imidazole concentrations (50, 100, 150, 250, and 500 mM) to optimize elution concentration and enhance
purity. A linear gradient, for example, using an FPLC system (see Steps 20–26), can improve separation from
contaminants further.

IMAC regeneration buffer (for resin regeneration in Steps 17–19 only) <R>
IMAC wash buffer <R>
Polyhistidine-tagged proteins with high binding affinities should remain bound to the column in the presence of
20 mM imidazole. As mentioned above, the amount of imidazole included in the binding and wash buffers should
be optimized for the specific protein of interest.

NaCl (2.0 M) (for resin regeneration in Steps 17–19 only)

Ni2+–NTA agarose (QIAGEN) or comparable IMAC chromatography resin (e.g., GE Healthcare;
IMAC Resin, Life Technologies; Ni2+–NTA agarose or HisLink, Promega)
Several different brands of resins, each with slightly different properties, are available for IMAC purification. Ni2+–
NTA resins are a popular starting point for IMAC purification. This protocol is for use with QIAGEN’s Ni2+–NTA
agarose resin.

NiSO4 (0.2 M) or other desired metal ion (for resin regeneration in Steps 17–19 only)
Polyacrylamide gel containing SDS
SDS (2%, w/v) (for resin regeneration in Steps 17–19 only)
SDS gel-loading buffer (5×) <R>
Mild detergents such as TWEEN 20, Triton X-100, or IGEPAL (formerly Nonidet P-40 or NP-40) may be included
to improve solubility and/or reduce nonspecific binding.

Stripping buffer (pH 7.0) (for resin regeneration in Steps 17–19 only) <R>

Equipment
ÄKTAFPLC System (GE Healthcare) or comparable system (e.g., BioLogic DuoFlow System; Bio-Rad)
(for FPLC in Steps 20–26 only)
As an alternative to an FPLC system, a simpler and significantly less expensive syringe or peristaltic pump may be
used to load, wash, and elute the polyhistidine fusion proteins from the prepacked HisTrap columns if a linear
elution gradient is replaced by a step elution.

Beckman JA20, Sorvall SS-34, or SA600 rotor or equivalent rotor with 40- to 50-mL centrifuge tubes
Dialysis tubing
Gravity-flow chromatography column (glass or polypropylene), with cap for bottom outlet
Prepacked Chelating HisTrap columns (GE Healthcare, 1 mL) or a comparable product (e.g., Bio-
Scale Mini Profinity IMAC Cartridges, 1 mL; Bio-Rad) (for FPLC in Steps 20–26 only)
The binding capacity of the precharged HisTrap columns (GE Healthcare) is unusually high among
IMAC cartridges (40 mg/mL resin). If nonspecific binding is problematic, a 1-mL Chelating HiTrap (GE
Healthcare; note difference in name) or 1-mL Mini Profinity IMAC Cartridge (Bio-Rad) may be more suitable
for medium-scale cultures because of their lower binding capacity (
15 mg/mL resin) and hence lower nonspe-
cific binding. However, HisTraps are used here because of their comparable cost, precharged format, and
significantly higher binding capacity. All of these column types can be regenerated and used for purification of

206 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

Purification of Polyhistidine-Tagged Proteins by IMAC

multiple batches of the same recombinant proteins. Separate columns should be used for each recombinant
protein.

Rocking platform or tube rotator (e.g., Thermo Scientific Labquake)

METHOD

The water and chemicals used for purification should be of a high purity and filtered through a 0.45-µm filter before use
to prevent contamination and column blockage. All buffers and samples should be used at 4˚C. High-purity imidazole
should have relatively low or no absorbance at 280 nm.
Before starting a purification procedure, conditions for expression should be optimized to yield the highest possible
amount of soluble protein. Estimations of the total amount of His-tagged protein present in the cell lysate can be used to
determine the amount of resin needed. Using too much IMAC resin allows host proteins to bind nonspecifically,
whereas using too little results in protein loss in the loading step.
Details of IMAC mechanics are presented in the Discussion section.

Preparation of the IMAC Resin

1. Gently invert the bottle of metal ion-charged Ni2+–NTA chromatography resin to mix the slurry,
and transfer 1 mL of the 50% (v/v) resin slurry to a tube. Centrifuge the resin slurry at 1000g for
10 sec. Carefully remove and discard the supernatant.
The amount of resin required to bind the desired amount of protein should be optimized. Ni2+–NTA IMAC
resins (QIAGEN) can bind 5–10 mg of protein per milliliter of resin, depending on the sample. The amount
of resin used should be the minimal amount required to bind the desired amount of protein. Using more resin
than is necessary can result in an increase in nonspecific binding, and using too little resin results in a loss of
protein. Resin binding capacities will vary based on the metal, chelator, and manufacturer. See Steps 17–19
for recharging instructions if the resin is not yet charged. The suggested volume of 1 mL of slurry is a starting
point for 1 L of E. coli cell growth.
2. Wash the resin by adding 1.5 mL (or 3 bed volumes of settled resin) of filtered distilled water and
mixing gently. Centrifuge the resin slurry at 1000g for 10 sec, and discard the supernatant.
One bed volume is equivalent to the volume of the settled bed of resin. For example, if 1 mL of 50% slurry is
added, then the bed volume is 0.5 mL. Therefore, the volumes used for washes and equilibration depend on
the amount of resin used.
3. Equilibrate the resin by adding 1.5 mL (or 3 bed volumes) of IMAC binding buffer. Mix the resin
with the buffer, then centrifuge the resin slurry at 1000g for 10 sec, and discard the supernatant.
The resin is now ready for use in Step 5.

Purification of the Polyhistidine-Tagged Protein

4. Prepare the cell lysate.
Protease inhibitors may be added at this step, but do not add EDTA or other chelators that will remove the
metal ions from the affinity resin, eliminating its ability to bind histidines.
5. Add the cleared lysate to the Ni2+–NTA resin prepared in Steps 1–3 and gently mix on a rocking
platform for 1 h at 4˚C.
6. Transfer the resin and bound protein to a clear polypropylene or glass chromatography column
with the bottom outlet capped. Allow the resin to pack under gravity flow, and ensure that no air
is trapped within the bed. Remove the cap and allow the lysate to flowthrough.
Prepare an aliquot of the cell lysate and flowthrough for SDS-PAGE analysis by mixing 15 µL with 5 µL of
5× SDS loading buffer and heating for 2 min on an 85˚C heat block; store this sample at −20˚C or on ice
until use.
Do not allow the resin to dry out. Perform all steps with chilled buffers and at 4˚C whenever possible. If
empty columns are not available, the remaining steps also may be performed using a batch procedure
similar to the one used in the section Preparation of the IMAC resin above.
Wash the column with 5 mL (or 10 bed volumes) of IMAC binding buffer.

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194 207

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

C.L. Kielkopf et al.

7. Wash the column with 5 mL (or 10 bed volumes) of IMAC binding buffer. Continue washing the
column until the A280 of the flowthrough ceases to decrease (optimally at <0.01 absorbance unit).
Use the binding buffer as a blank because imidazole absorbs at 280 nm to an extent depending on
its purity and concentration.
8. Wash with 1.5 mL (or 3 bed volumes) of IMAC wash buffer.
The A280 of the flowthrough from the wash also is expected to plateau before elution. Keep the wash
flowthrough until analysis by SDS-PAGE confirms the location of the target protein.
Prepare an aliquot of the wash for SDS-PAGE analysis by mixing 15 µL with 5 µL of 5× SDS loading buffer
and heating for 2 min on an 85˚C heat block; store this sample at –20˚C or on ice until use.

Elution of the Polyhistidine-Tagged Protein

9. Elute the bound protein with 2 mL (or 4 bed volumes) of IMAC imidazole elution buffer. Collect
a 0.5-mL (1 bed volume) fraction from the column, then cap the column and let it sit for 10 min.
Repeat three times and monitor the A280 of each fraction.
Alternatively, the protein may be eluted with increasing imidazole concentration step gradients (50, 100,
250, and 500 mM) to optimize the imidazole concentration and increase purity. Most His-tagged proteins
will elute between 100 mM and 250 mM imidazole, but the exact concentration required is protein-depen-
dent. Instead of using imidazole, the bound protein can also be eluted using buffers of decreasing pH.
If a specific protease site is engineered between the tag and the target protein, on-column cleavage is an
alternative means for elution.
Prepare an aliquot of each eluate fraction for SDS-PAGE analysis by mixing 15 μL with 5 μL of 5× SDS loading
buffer and heating for 2 min on an 85˚C heat block; store this sample at −20˚C or on ice until use.
10. Briefly rewarm the SDS-PAGE samples to 85˚C, and then load each sample on a suitable
percentage SDS–polyacrylamide gel. The purified protein should be visible on the gel following
staining with Coomassie Brilliant Blue. The eluted fractions may be combined or, if a more
concentrated sample is desired, kept separate.
In some instances, a single IMAC purification step may not yield protein of sufficient purity. If this is the case,
a second IMAC purification step may be used following the cleavage of the polyhistidine tag from the target
protein by a specific protease. If the fusion protein lacks a specific protease site, another purification strategy
(e.g., ion exchange or size-exclusion chromatography) must be used.
11. If the polyhistidine tag is separated from the target protein by a specific protease site, add the
appropriate protease. Dialyze the sample overnight at 4˚C against a large volume of IMAC
binding buffer (also used in Step 3) to remove excess imidazole (e.g., 500 mL of dialysis buffer
for
2 mL of eluate, or a >250:1 [v/v] ratio). The amount of imidazole present in the binding
buffer may need to be optimized as described in the Reagents list above.
Dialysis selectively dilutes the components of a solution that are smaller than the pore size or molecular
weight cutoff (MWCO) of the dialysis membrane. These semipermeable membranes can be purchased as
tubing or cassettes made from regenerated cellulose or cellophane in a broad range of sizes (1–50 kDa). An
appropriately selected membrane will prevent the passage of the protein of interest (with a molecular-
weight cutoff [MWCO] that is at least half the size of the protein) while allowing for smaller molecules to
diffuse freely across the membrane.
The amount of protease to use and the conditions of the dialysis buffer will need to be adjusted according to
the specific protease and fusion protein. Cleavage while the fusion protein is still attached to the resin is an
alternative strategy.
12. Equilibrate the Ni2+–NTA resin from Step 9 with 3 bed volumes of fresh Wash buffer 2. Mix the
resin with the buffer, then centrifuge the resin slurry at 1000g for 10 sec, and discard the
supernatant. Repeat two more times (a total of three times) to remove any nonspecifically
bound proteins and residual imidazole.
The spent dialysis buffer should not be used to equilibrate the resin because it is contaminated with low
concentrations of imidazole that may interfere with binding of the cleaved tag, tagged protease, and
contaminants.

208 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

Purification of Polyhistidine-Tagged Proteins by IMAC

The Ni2+–NTA resin from Step 9 does not need to be regenerated before use but may be washed with 0.5 M
imidazole/0.5 M NaCl before equilibration in Wash buffer 2.
13. Add the dialyzed, cleaved protein to the resin bed. Gently mix on a rocking platform for 1 h at
4˚C.
14. Transfer the resin and bound protein to a clear polypropylene or glass chromatography column
with the bottom outlet capped. Allow the resin to pack under gravity, and ensure that no air is
trapped within the bed. Remove the cap and collect the flowthrough, which should contain the
target protein.
15. Wash the column with 3 bed volumes of Wash buffer 2. Collect fractions with 1 bed volume each.
Prepare aliquots of the flowthrough and wash fractions for SDS-PAGE analysis by mixing 15 µL with 5 µL of
5× SDS loading buffer and heating for 2 min on an 85˚C heat block; store this sample at −20˚C or on ice
until use.
16. Briefly rewarm the samples to 85˚C, and then load each sample on a suitable percentage SDS–
polyacrylamide gel. The purified protein should be visible on the gel following staining with
Coomassie Brilliant Blue.
The fractions may be combined with the flowthrough; if a more concentrated sample is desired, kept them
separate. To store the purified protein in an active form, see Box 1.

Regenerating and Cleaning the Ni2+–NTA Resin

The Ni2+–NTA resin may be reused many times for the purification of a specific recombinant protein. To prevent
contamination, a new batch of resin should be used for each recombinant protein. Between runs, the resin should be
washed with 0.5 M NaOH for 30 min, followed by water, and stored in 20% ethanol to prevent microbial growth. If the
column becomes grayish or otherwise loses the characteristic light-blue color of chelated Ni2+ ions, a regeneration
procedure should be followed. In most instances, simply washing the column with IMAC imidazole elution buffer
followed by the Stripping buffer (Step 17) and recharging (Step 19) is sufficient. The extensive IMAC washing
procedure provided below can be used in more extreme cases (e.g., if the column becomes clogged or protein is
precipitated on the column). In this procedure, a “volume” is equivalent to the volume of the bed of resin that is being
cleaned. For example, if 0.5 mL of resin is being cleaned, then 1 volume is equal to 0.5 mL. This procedure is
appropriate for use with most Ni2+-charged resins. For Co2+-charged and/or CM-Asp resins, see the manufacturer’s
instructions for modifications.

17. Strip a batch of resin by adding the following solutions sequentially with gentle mixing at each
step. No incubation period is required for these steps. Exchange the buffers by centrifuging the
resin in a tube at 1000g for 10 sec, decanting the supernatant, and adding the next buffer. The
amounts suggested below are for the 0.5-mL bed volume used in the main protocol above.

Stripping buffer 2.5 mL (or 5 volumes)

NaCl (2.0 M) 1.5 mL (or 3 volumes)
Water 1.5 mL (or 3 volumes)
18. If necessary, clean the resin successively with the solutions listed below. Gently mix the resin at
each step, and exchange the buffer by centrifuging the resin at 1000g for 10 sec.

IMAC regeneration buffer (to remove bound 1 mL (or 2 volumes)

protein)
Water 2.5 mL (or 5 volumes)
SDS (2%) 1.5 mL (or 3 volumes)
Ethanol (25%) 0.5 mL (or 1 volume)
Ethanol (50%) 0.5 mL (or 1 volume)
Ethanol (75%) 0.5 mL (or 1 volume)
Ethanol (100%) 2.5 mL (or 5 volumes)
Ethanol (75%) 0.5 mL (or 1 volume)
Ethanol (50%) 0.5 mL (or 1 volume)
Ethanol (25%) 0.5 mL (or 1 volume)
Wash extensively with water

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194 209

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

C.L. Kielkopf et al.

BOX 1. MAINTAINING PROTEIN QUALITY DURING STORAGE

The extraction and purification of proteins remove them from their native environment, exposing them to
potentially harmful conditions that can result in denaturation, aggregation, or a loss of activity (discussed in
Deutscher 2009). Buffer and temperature conditions are important elements in the storage of proteins.

Buffer Conditions
Keeping the buffer conditions as close to the native state as possible while removing damaging molecules
(such as proteases) often helps to preserve the integrity of the protein during storage. For example, soluble
cytosolic proteins are normally produced in relatively low salt concentrations, at a near-neutral pH, under
reducing conditions, and in the presence of a high concentration of macromolecules. Taking this into
consideration, optimal protein storage buffers will often include salts (NaCl, KCl, etc.; 50–300 mM), a
buffer (Tris, HEPES, etc.; 25 mM), reducing agents (dithiothreitol or β-mercaptoethanol; 1–5 mM), protease
inhibitors (as needed), and a chelating agent (such as EDTA; 1–5 mM) to inhibit metal-induced oxidation and
metalloproteases. Each one of these variables can have an effect on the stability of the native state and on
their unfolding/refolding rates. Mimicking the exact native conditions is not always straightforward in vitro,
particularly when it comes to reproducing the molecular crowding effect (caused by the high concentration
of macromolecules), which contributes to thermal stability (Despa et al. 2005). Perhaps the easiest way to
imitate molecular crowding is to keep the protein concentration high (above 1 mg/mL) during both purifi-
cation and storage. In situations in which this is not possible, additives (i.e., polymers) can be used to the
same effect (Bhat and Timasheff 1992; Parsegian et al. 2000; Timasheff 2002). Alternatively, protein-stabi-
lizing cocktails containing proprietary mixes of stabilizing compounds are available as kits (Thermo Scien-
tific Pierce, Expedeon, etc.).

Temperature Conditions
Much like the storage buffer variations, temperature requirements are also protein-specific. In general,
proteins are best stored at or below 4˚C in sealed, autoclaved glassware or polypropylene tubes. Storing
proteins at room temperature can lead to protein degradation, inactivation, or microbial growth. Partic-
ularly stable proteins may be kept at 4˚C for short-term storage (a day to a couple weeks) in a simple
storage buffer with relatively few adverse effects. At this temperature, the sample is more easily dispensed
as needed without thawing and refreezing the sample, but may also require the addition of antimicrobial
agents, such as sodium azide (0.02%–0.05%, w/v), to prevent contamination. For more long-term
storage, or less stable proteins, reducing the storage temperature (to −20˚C or −80˚C) is highly
recommended.
Storage at freezing temperatures is common for protein samples and can significantly increase their
longevity. Depending on the stability of the protein, it may be advisable to include 5%–15% glycerol as a
cryoprotectant. Alternatively, the protein of highly concentrated solutions (>10 mg/mL) often serves as its
own cryoprotectant. There are a few different ways to prepare a protein for storage at subzero temperatures.
(We routinely keep 5%–15% glycerol present for freezing cryoprotectant.) One of the most common
methods is to freeze the protein dropwise (
100 µL) in liquid nitrogen and transfer the frozen drops to
storage tubes while still under liquid nitrogen. This method allows for rapid protein freezing and reduces
the formation of ice crystals that can denature proteins. Another recently reported method (Deng et al.
2004) claims that protein samples frozen in small aliquots in PCR tubes freeze faster and suffer less damage
to the sample than when the drop-freezing method is used. In either case, the protein should be stored in
small, single-use volumes to avoid the need to refreeze any unused protein because freeze–thaw cycles
have been shown to decrease protein activity (Nema and Avis 1993). Alternatively, the addition of 50%
glycerol or ethylene glycol can prevent freezing at –20˚C, allowing for easy use without thawing
the sample.

19. Recharge the resin with 1 mL (or 2 volumes) of 0.2 M NiSO4, wash with 1.5 mL (or 3 volumes) of
H2O, and equilibrate with the appropriate chromatographic buffer. Store the resin in 20%
ethanol.
The column should be white after stripping with EDTA (or after elution of protein with EDTA) and should
return to a pale-blue color after regeneration if using Ni2+.

210 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

Purification of Polyhistidine-Tagged Proteins by IMAC

Fast Performance Liquid Chromatography Purification of Histidine-Tagged Proteins

The following procedure is modified from that recommended for the ÄKTA System (GE Healthcare) and their pre-
packed 1-mL HisTrap column charged with Ni2+, but may also be applied to other systems. A “column volume” refers
to the volume of packed resin contained in a particular column (1 mL in this case). (This procedure is adapted from GE
Healthcare Instructions 11-0008-88 AF HisTrap Affinity Columns.)

20. Fill the connection tubing with water, and connect the column “drop-to-drop” to avoid intro-
ducing air into the system.
When connecting tubing and columns, be sure that there is no air in the ends by pushing liquid through the
system while connecting. A drop of liquid from each end should be present outside of the connections upon
connection, hence, “drop-to-drop.”
21. Wash the column with 5 mL (or 5 column volumes) of water to remove the storage buffer (20%
ethanol).
Follow the manufacturers’ guidelines for pressure and flow rate limits, because these will vary between
products. For the 1-mL HisTrap (GE Healthcare), the column pressure limit is 0.3 MPa (3 bar, 43.5 psi), and
the recommended flow rate is 1 mL/min.
22. Equilibrate the column with 5 mL (or 5 column volumes) of IMAC binding buffer.
23. Prepare the cell lysate.
24. Apply the pretreated cell lysate (containing up to 40 mg of target protein for a 1-mL HisTrap)
using a peristaltic pump, superloop, or syringe (depending on the setup).
Refer to the manufacturer’s guidelines when determining the binding capacity of the columns because these
vary between manufacturers. If the capacity of the column is likely to be exceeded, then multiple 1-mL
columns can be connected in tandem or replaced with a 5-mL column to optimize the yield. Note that using
columns in tandem is expected to increase the back pressure.
25. Wash with IMAC binding buffer until the absorbance at 280 nm reaches a stable baseline. Elute
the polyhistidine-tagged protein with IMAC imidazole elution buffer using a linear gradient up to
100% over 20 column volumes.
26. Pool the eluted protein based on the A280 and SDS-PAGE analysis of the fractions.
At this point, the protein may be used as a fusion protein, or the 6xHis tag may be removed if a specific
protease site is present in the fusion protein. If the polyhistidine tag is to be cleaved from the target protein,
continue in an analogous manner to Steps 11–16 for a second pass through the IMAC cartridge.

TROUBLESHOOTING

Problem: Histidine-tagged protein does not bind to the IMAC resin.

Solutions:
• Ensure that the sample and buffer components do not interfere with His-tag binding. Concen-
trations of strong reducing agents (DTT, DTE, TCEP) and chelating agents (EDTA, EGTA) in
all buffers should be within allowable limits. Consult the manufacturer’s manual for a list of
compatible chemicals and suggested concentrations. Resin-bound metal that has been reduced
will typically change color (Ni2+ will turn from blue to brown), and stripping of metal ions by a
chelating agent will turn the resin white. Sample and loading/wash buffers should have a neutral or
slightly alkaline pH (pH 7−8) to limit His-tag protonation.
• The His-tag may be obstructed by intra- or intermolecular interactions. Moving the tag to the
other terminus or purifying the protein under denaturing (or partially denaturing) conditions may
improve accessibility. Modifying the polyhistidine tag by adding up to 12 histidines or by inserting
a linker between the tag and the protein may also improve binding efficiency.
• The concentration of imidazole in the sample, binding, or wash buffers may be too high. Reduce
the imidazole concentration to a level that allows binding of the target protein to the IMAC resin.
• Not all resins are provided in the metal ion-bound form. Confirm that the IMAC resin has
been charged with metal ion and prepared as described in Steps 17–19. Once properly charged,

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194 211

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

C.L. Kielkopf et al.

the column should retain the hue of the original ion salt solution (blue for nickel, pink for
cobalt, etc.).

Problem: The His-tagged protein of interest binds to the resin, but more than one protein elutes in
the peak.
Solutions:
• Contaminating proteins may have affinity for the IMAC resin and coelute with the His-tagged
protein. If the His-tagged protein has a higher relative affinity, it will elute at a higher concentra-
tion of imidazole. Eluting with a shallow (≥20 column volumes) linear or stepwise gradient of
elution buffer can be used to determine an intermediate concentration of imidazole that removes
contaminants without eluting the target protein.
• If contaminating proteins cannot be removed by optimizing the imidazole concentrations in the
binding and elution buffers, further purification steps may be required. Commonly used proce-
dures include ion-exchange chromatography and/or gel filtration chromatography.
• Proteolytic degradation products may retain the His tag and copurify with the full-length protein.
Use protease inhibitors.

DISCUSSION

Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for
divalent metal cations (usually Ni2+) immobilized as transition metal chelate complexes on a chro-
matography resin (for reviews, see Bornhorst and Falke 2000; Block et al. 2009). Because few natural
proteins bind to immobilized divalent metal ions with significant affinities, recombinant polyhisti-
dine-labeled proteins can be purified to near homogeneity, often in a single step, using metal chelate
affinity chromatography. Wash steps remove the majority of contaminating proteins that bind the
resin, and the protein of interest is eluted with a soluble competing chelator. General considerations
during IMAC include accessibility of the polyhistidine tag, type of metal ion and chelating matrix,
and the pH and buffer components for binding, wash, and elution steps. Metal affinity chromatog-
raphy is popular because it is both highly effective and relatively insensitive to protein folding, ionic
strength, chaotropes, and detergents. Because of its high binding capacity, up to 50 mg of recombinant
protein can be purified using only 1 mL of IMAC resin (2.5 µmol of a 20-kDa protein using Ni2+–
NTA agarose; QIAGEN). Considering the high efficiency, capacity, concentrating power, and speed
of this technique, it is often the first (and sometimes only) strategy used when developing a
purification protocol.

Polyhistidine Sequences
The affinity of transition metals (Zn2+, Cu2+, Co2+, and Ni2+, etc.) for specific amino acids (histidine
and cysteine) was noted as early as 1948 (Hearon 1948). But it was not until 1975 that immobilized
metal substrates were first exploited for the purification of proteins carrying surface-exposed histidine
residues (Porath et al. 1975). Today, a protein of interest is often modified to contain a polyhistidine
sequence in an exposed and relatively flexible site, most frequently at the amino or carboxyl terminus
of the protein (Van Reeth et al. 1998). To increase accessibility, one or two glycine residues are placed
between the polyhistidine tract and the remainder of the protein sequence. Many commonly used
plasmids (some versions of pcDNA vectors, Life Technologies; pET vectors, Novagen; and pQE
vectors, QIAGEN) contain polyhistidine sequences that are composed of six tandem histidine residues
(6×His tag). If the 6×His tag is occluded by the protein fold, then either lengthening (8×His, 10×His,
12×His) or moving the tag to the opposite terminus of the protein may enhance accessibility and
binding to IMAC resin (Heijbel 2003; Mohanty and Wiener 2004). Lengthened polyhistidine tags may

212 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

Purification of Polyhistidine-Tagged Proteins by IMAC

also increase the affinity of the fusion protein for the immobilized metal ions, so that more stringent
imidazole washes can be used to remove contaminating proteins before elution of the target protein.

Imidazole and Elution Methods

During binding of the target protein to the resin, low concentrations of imidazole are usually included
to reduce nonspecific interactions (5–30 mM imidazole, depending on the metal ion affinity of the
target protein). Usually the ionic strengths of the IMAC buffers are kept relatively high (0.2–1.0 M
NaCl) to reduce nonspecific electrostatic interactions between contaminants and the metal ions. The
pH of the binding and wash buffers is 7–8 to maintain the histidines in an unprotonated state
appropriate for metal chelation. Certainly, the buffers used for the washing and binding steps
should not contain strong reducing agents (DTT) or chelators (EDTA or citrate), which would
remove the immobilized metal ions.
The target protein usually is eluted by competition with high concentrations of imidazole.
Alternative methods for elution include stripping the metal ions using chelators such as EDTA
or decreasing the pH of the solution 1–2 units below the pKa of the histidine side chain (
6.0).
Imidazole is the most selective and gentle of these eluents and should be used whenever possible.
Elution buffers commonly contain between 100 and 250 mM imidazole at pH 7–8 for Ni2+–NTA
resin. The lowest effective concentration can be established by a linear gradient of 10–250 mM
imidazole. Although EDTA is more efficient than imidazole, it also carries the immobilized metal
from the resin in addition to the protein of interest. Some applications may require that the
metal ions be removed from the sample, either by dialysis or by a second chromatographic step.
For example, Ni2+ ions and complexes have been shown to be toxic and to interfere with the
activity of some enzymes (particularly iron-dependent enzymes) (Chen et al. 2005, 2010). Alterna-
tively, a different metal species can be used when a metal ion interferes with protein activity or
downstream applications. For example, immobilized Zn2+ ions are preferable for zinc fingers and
other proteins that depend on zinc for structure or activity, because metal ions partially exchange on
the chromatographic resin despite the relatively high affinities of tetradentate ligands (Block et al.
2009).
Because unique protease cleavage sites commonly are inserted between the fusion tag and protein
of interest, proteases can be used to elute proteins from the affinity matrix and to remove 6×His
sequence during purification (Wen-Hui and Howard 2010). Several of these proteases also contain a
6×His modification (in this case, uncleavable) that allows the protease to be removed from the cleaved
target protein by a second IMAC purification step. In addition to the polyhistidine-tagged protease,
this second IMAC purification step also removes uncut fusion proteins, the polyhistidine tag, and any
contaminating proteins that may have bound the resin during the first purification step (Arnau et al.
2006).

Chelating Resins and Ligands

Most chelating resins are supplied free of metal ions and need to be charged with a suitable ion before
use. The type of ion depends on the application and the specific characteristics of the protein of
interest, such as the length and position of the affinity tag and the pH of the buffers. The Lewis metal
ions (Ni2+, Co2+, Zn2+, and Cu2+) are most common; Ni2+ is used frequently because of its high
affinity for polyhistidines and greater aqueous solubility. As a consequence of its higher affinity
compared with Co2+, Ni2+ unfortunately also binds contaminant proteins more efficiently. Accord-
ingly, the purity of low-abundance polyhistidine-tagged proteins may benefit from use of the less-
common Co2+ ions. However, the amount of recovered target protein may decrease as well. Because of
the lower affinity for immobilized Co2+ ions compared with the Ni2+ counterpart, lower concentra-
tions of imidazole (150–200 mM) are usually sufficient for elution of the target protein from resins
charged with Co2+.
Following early work using immobilized metal ions to purify proteins based on their natural
histidine content (Porath et al. 1975), a variety of improved chelating ligands have been developed

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194 213

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

C.L. Kielkopf et al.

(Fig. 1; for review, see Block et al. 2009). Iminodiacetic acid (IDA) was the chelating ligand
originally used by Porath’s group (Porath et al. 1975) and remains available in several commercial
IMAC resins (including resins from GE Healthcare, Pierce, and USB Affymetrix). In the late 1980s,
the improved nitriloacetic acid (NTA) chelating ligand was developed (Fig. 1; Hochuli et al. 1987)
and commercialized by QIAGEN for purification of polyhistidine fusion proteins. Subsequently, a
carboxymethyl-aspartate ligand (CM-Asp, also called TALON, by Clontech) has become widely
used with Co2+ ions (Chaga et al. 1999). The IDA ligand has a tridentate coordination of the
bound metal ions that differs from the tetradentate coordination by the NTA and CM-Asp
ligands. These coordination schemes, respectively, leave three or two sites available for coordination
of histidines. The tridentate IDA ligand suffers from higher leaching of metal ions to the surround-
ing buffer (Hochuli 1989). The purity of the tagged protein may be lower following use of IDA
compared with tetradentate ligands, possibly because of nonspecific electrostatic interactions of
contaminating proteins with IDA sites that have lost their bound metal ion. A pentacoordinate
tris(carboxymethyl)ethylenediamine (TED) ligand (including resins from MACHEREY-NAGEL)
with very low metal ion leaching is a third option. However, the availability of only a single
empty site for histidine coordination reduces the capacity of TED to bind polyhistidine-tagged
proteins. Accordingly, the IDA or NTA resins charged with Ni2+ ions are the most commonly
used matrices for proteins tagged with polyhistidine residues.

A Protein B Protein

N N N N N N
N N N N N N
N N N N N N
N N N N N N

Ni2+
O Ni2+ O Ni-NTA
O O Ni-IDA O
CH2
O
O N CH2
O O N
CH2 CH2
CH
O

C Protein

N N N
N N N
N N N
N N N

O O O O
Ni-TED
CH2 Ni2+
CH2
N CH2 N
CH2
CH2 O

FIGURE 1. IMAC resin. Shown are the interactions between a metal ion (Ni2+), the chelating groups of three commonly
used substrate-bound chelators, and the polyhistidine tag of a fusion protein. (A) Iminodiacetic acid (IDA) is in a
tridentate coordination mode leaving three coordination sites available to interact with the polyhistidine tag (top). (B)
Nitriloacetic acid (NTA) is in a tetradentate coordination mode with the metal ion providing two open sites for
polyhistidine binding. (C ) Tris(carboxymethyl)ethylenediamine (TED) is in a pentadentate coordination mode allow-
ing only one valency to bind histidine.

214 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

Purification of Polyhistidine-Tagged Proteins by IMAC

RECIPES

Dialysis Buffer and Wash Buffer 2

Reagent Quantity (for 1 L) Final concentration

Sodium phosphate 0.57 g of sodium phosphate monobasic, 25 mM
(pH 7.4) anhydrous and 2.88 g of sodium
phosphate dibasic, anhydrous
NaCl 8.76 g 150 mM
Store at 4˚C.

IMAC Binding Buffer for Protein Purification

Reagent Quantity (for 1 L) Final concentration

Sodium phosphate 1.13 g of sodium phosphate 50 mM
(pH 7.4) monobasic, anhydrous and 5.75 g of
sodium phosphate dibasic, anhydrous
NaCl 29.22 g 500 mM
Imidazole (pH 8.0) 0.68 g 10 mM
Store at 4˚C.

IMAC Imidazole Elution Buffer

Reagent Quantity (for 1 L) Final concentration

Sodium phosphate 1.13 g of sodium phosphate monobasic, 50 mM
(pH 7.4) anhydrous and 5.75 g of sodium phosphate
dibasic, anhydrous
NaCl 29.22 g 500 mM
Imidazole (250 mM) 17.0 g 250 mM
Store at 4˚C.

IMAC Regeneration Buffer

Reagent Quantity (for 100 mL) Final concentration

Guanidinium chloride 57.3 g 6M
Acetic acid, glacial (17.5 M) 1.14 mL 0.2 M
Store at room temperature.

IMAC Wash Buffer

Reagent Quantity (for 1 L) Final concentration

Sodium phosphate 1.13 g of sodium phosphate monobasic, 50 mM
(pH 7.4) anhydrous and 5.75 g of sodium phosphate
dibasic, anhydrous
NaCl 29.22 g 500 mM
Imidazole (pH 8.0) 1.36 g 20 mM
Store at 4˚C.

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194 215

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press
C.L. Kielkopf et al.
SDS Gel-Loading Buffer (5×)
Reagent
Tris-Cl (1 M, pH 6.8)
SDS (electrophoresis grade)
Bromophenol blue
Glycerol (80%, v/v)
Dithiothreitol (1 M)
H2O
SDS gel-loading buffer (5×) lacking DTT can be stored at room temperature. Add DTT from a 1 M
stock just before the buffer is used.
Stripping Buffer (pH 7.0)
Reagent Quantity (for 100 mL)
EDTA (0.5 M, pH 8.0)
NaCl
Store at 4˚C.
REFERENCES
Arnau J, Lauritzen C, Petersen GE, Pedersen J. 2006. Current strategies for
the use of affinity tags and tag removal for the purification of recom-
binant proteins. Protein Expr Purif 48: 1–13.
Bhat R, Timasheff SN. 1992. Steric exclusion is the principal source of the
preferential hydration of proteins in the presence of polyethylene
glycols. Protein Sci 1: 1133–1143.
Block H, Maertens B, Spriestersbach A, Brinker N, Kubicek J, Fabis R,
Labahn J, Schafer F. 2009. Immobilized-metal affinity chromatography
(IMAC): A review. Methods Enzymol 463: 439–473.
Bornhorst JA, Falke JJ. 2000. Purification of proteins using polyhistidine
affinity tags. Methods Enzymol 326: 245–254.
Chaga G, Hopp J, Nelson P. 1999. Immobilized metal ion affinity chroma-
tography on Co2+-carboxymethylaspartate-agarose Superflow, as dem-
onstrated by one-step purification of lactate dehydrogenase from
chicken breast muscle. Biotechnol Appl Biochem 29: 19–24.
Chen H, Davidson T, Singleton S, Garrick MD, Costa M. 2005. Nickel de-
creases cellular iron level and converts cytosolic aconitase to iron-regu-
latory protein 1 in A549 cells. Toxicol Appl Pharmacol 206: 275–287.
Chen H, Giri NC, Zhang R, Yamane K, Zhang Y, Maroney M, Costa M.
2010. Nickel ions inhibit histone demethylase JMJD1A and DNA repair
enzyme ABH2 by replacing the ferrous iron in the catalytic centers.
J Biol Chem 285: 7374–7383.
Deng J, Davies DR, Wisedchaisri G, Wu M, Hol WG, Mehlin C. 2004. An
improved protocol for rapid freezing of protein samples for long-term
storage. Acta Crystallogr D Biol Crystallogr 60: 203–204.
Despa F, Orgill DP, Lee RC. 2005. Molecular crowding effects on protein
stability. Ann N Y Acad Sci 1066: 54–66.
Deutscher MP. 2009. Maintaining protein stability. Methods Enzymol 463:
121–127.
Hearon JZ. 1948. The configuration of cobaltodihistidine and oxy-bis
(cobaltodihistidine). J Natl Cancer Inst 9: 1–11.
216
Quantity (for 1 mL) Final concentration
0.25 mL 250 mM
80 mg 8%
1 mg 0.1%
0.5 mL 40% (v/v)
0.1 mL 100 mM
0.25 mL
Final concentration
40 mL 0.2 M
2.92 g 500 mM
Heijbel A. 2003. Purification of a protein tagged with (His)6 at its N-terminus,
C-terminus, and both N- and C-termini using different metal ions. Life
Science News–Amersham Biosciences AB, Uppsala, Sweden.
Hochuli E. 1989. Genetically designed affinity chromatography using a novel
metal chelate absorbent. In Biologically active molecules: Identification,
characterization, and synthesis: Proceedings of a Seminar on Chemistry on
Biologically Active Compounds and Modern Analytical Methods (ed.
Schlunegger UP), Vol. 411, pp. 217–239. Springer-Verlag, Berlin.
Hochuli E, Dobeli H, Schacher A. 1987. New metal chelate adsorbent selec-
tive for proteins and peptides containing neighbouring histidine resi-
dues. J Chromatogr 411: 177–184.
Mohanty AK, Wiener MC. 2004. Membrane protein expression and pro-
duction: Effects of polyhistidine tag length and position. Protein Expr
Purif 33: 311–325.
Nema S, Avis KE. 1993. Freeze–thaw studies of a model protein, lactate
dehydrogenase, in the presence of cryoprotectants. J Parenter Sci
Technol 47: 76–83.
Parsegian VA, Rand RP, Rau DC. 2000. Osmotic stress, crowding, prefer-
ential hydration, and binding: A comparison of perspectives. Proc Natl
Acad Sci 97: 3987–3992.
Porath J, Carlsson J, Olsson I, Belfrage G. 1975. Metal chelate affinity chro-
matography, a new approach to protein fractionation. Nature 258:
598–599.
Timasheff SN. 2002. Protein hydration, thermodynamic binding, and pref-
erential hydration. Biochemistry 41: 13473–13482.
Van Reeth T, Dreze PL, Szpirer J, Szpirer C, Gabant P. 1998. Positive selec-
tion vectors to generate fused genes for the expression of his-tagged
proteins. BioTechniques 25: 898–904.
Wen-Hui KK, Howard CA. 2010. Absorptive detagging of poly-histidine
tagged protein using hexa-histidine tagged exopeptidase. J Chromatogr
A 1217: 7749–7758.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot102194
 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 2, 2020 - Published by
Cold Spring Harbor Laboratory Press

Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity

Chromatography
Clara L. Kielkopf, William Bauer and Ina L. Urbatsch

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot102194

Email Alerting Receive free email alerts when new articles cite this article - click here.
Service

Subject Browse articles on similar topics from Cold Spring Harbor Protocols.
Categories
Affinity and Immunoaffinity Chromatography (55 articles)
Chromatography (42 articles)
Chromatography, general (132 articles)
Protein Expression and Interactions (85 articles)
Protein Identification and Analysis (194 articles)
Proteins and Proteomics, general (562 articles)
Tagging Proteins (55 articles)

To subscribe to Cold Spring Harbor Protocols go to:

http://cshprotocols.cshlp.org/subscriptions

© 2020 Cold Spring Harbor Laboratory Press