Scientific & Technical Report
PN 33354
IMAC Purification of Polyhistidine-tagged Protein
Using the AcroPrep™ 96 Filter Plate
By Tao Hu, Ph.D. and Kevin Seeley, Ph.D., Pall Corporation
Abstract
Bottlenecks in protein sample preparation
highlight the need to optimize screening strategies
and develop more effective automated systems
to recover proteins for proteomic analysis. Fully
automated purification strategies may include
a number of steps where lysate clearance
(AcroPrep 96 prefiltration plates), desalting and
protein concentration (AcroPrep 96 ultrafiltration
plates) are needed. Beyond standard sample
preparation steps, optimizing the purification
for over-expressed tagged-recombinant proteins
requires the screening of a collection of subclones
that often have highly variable expression and
activity levels. Although size-separation applications can be done effectively using ultrafiltration, a
more specific affinity Immobilized Metal Affinity
Chromatography (IMAC) system is typically
needed to purify biomolecules from crude lysates.
This study demonstrates the use of AcroPrep 96
filter plates containing microporous membranes for
the preparation of recombinant proteins isolated
from IMAC resin minicolumns. The versatility of
the AcroPrep 96 filter plate is demonstrated by
the creation of a matrix of IMAC conditions within
a single plate. The ability to create a screening
matrix assists in the selection of an effective
ligand for a target protein and allows the design
of parallel processing tests to optimize resin to
lysate ratios for a particular clone. The construction
of the AcroPrep 96 filter plate allows both manual
and automated handling, demonstrates bead
retention, and shows consistent well-to-well
performance, effectively giving the protein
biochemist an edge in the development of
protein purification protocols.
Background
Immobilized Metal Chelating
Chromatography
Immobilized Metal Affinity Chromatography (IMAC)
(1,2) is a robust method for purifying polyhistidinetagged recombinant proteins. This is achieved by
using the natural tendency of histidine to form a
complex with divalent metals around neutral pH.
Fusion with the histidine peptides is now one of
the most commonly used techniques for affinity
purification. Immobilizing the metal ion on a
chromatographic resin by chelation allows the
separation of the histidine-tagged proteins from
most untagged proteins even under denaturing
conditions. The binding interaction with the
tagged protein is pH dependent. The bound
sample can be eluted from the resin by reducing
the pH and increasing the ionic strength of the
buffer or by including EDTA or imidazole in the
buffer. Since target protein can be purified from
a large volume of crude lysate in a single step,
IMAC purification offers significant time savings
over less selective multi-step procedures. Like
any other type of chromatography, parameters
of purification conditions need to be optimized
for the successful practice of any IMAC strategy.
These parameters include the choice of metal ion
and resin type, the amount of resin to be used,
and the condition of binding and elution buffers.
AcroPrepTM 96 Filter Plate
Increasing demand of protein sample preparation in proteomics
accentuates the need to develop more effective strategies
for high throughput protein purification. Fully automated
purification strategies may include a number of steps where
lysate clearance, desalting and protein concentration are
needed (3, 4, 5). Beyond standard sample preparation
steps, selection of the most suitable protein construct for
downstream applications requires the screening of a collection
of subclones that often have highly variable expression and
activity levels. Performing IMAC on a multi-well filter plate
platform is a fast and simple way to purify multiple histidinetagged proteins in parallel. It allows the high throughput
screening of different protein expression constructs. In addition,
it facilitates the optimization of various purification parameters
including the choice of metal ion resin, the sample-to-resin
ratio, and the elution condition.
Vacuum manifold (Pall Life Sciences)
The performance of the AcroPrep filter plates is optimized
when used on the Pall Life Sciences vacuum manifold
(PN 5017). Other ANSI/SBS x. 2004 compatible
manifolds can be used with AcroPrep filter plates.
Reagents
1. Polyhistidine-tagged TEV protease construct and human
Brf2 truncation construct are provided courtesy of
Dr. Joshua-Tor’s lab in Cold Spring Harbor Laboratories
(Cold Spring Harbor, NY)
2. HiTrap* Chelating Sepharose HP (Amersham Biosciences,
Uppsala, Sweden)
3. Ni-NTA* Superflow (Qiagen, Valencia, CA)
4. TALON* Metal Affinity Resin (BD Bioscience, Palo Alto, CA)
5. Buffer for purification under native condition:
• Washing Buffer: 100 mM NaPi, 300 mM NaCl, 20 mM
Imidazole, pH 8.0
• Elution Buffer: 100 mM NaPi, 300 mM NaCl, 250 mM
Imidazole, pH 8.0
6. Buffer for purification under denaturing condition:
• Washing Buffer: 100 mM NaPi, 6M Urea, pH 6.3
• Elution Buffer: 100 mM NaPi, 6M urea, pH 3.0, 4.0 or 5.0
Step-by-step Procedures
Set-up
1. Connect the vacuum manifold to a liquid trap.
The AcroPrep 96 filter plate was designed to fit Society for
Biomolecular Screening (SBS) compatible manual manifolds
as well as robotic equipment. Combined with immobilized
metal affinity resin, the plate can be used to purify small
amounts of recombinant proteins engineered with a
polyhistidine tag. It is ideal for developing protocols
for high throughput processing of protein purification.
Specific advantages of using AcroPrep 96 filter plates are:
2. Connect the liquid trap to a vacuum source through a
Vacushield™ vent filter (PN 4402).
3. Place the collection plate and appropriate spacer block
in the lower chamber of the Pall Life Sciences vacuum
manifold (PN 5017).
4. Replace the upper chamber of the vacuum manifold.
• Polypropylene construction is chemically resistant and
biologically inert
5. Place the AcroPrep 96 filter plate on the gasket located on
the top chamber of the vacuum manifold. Ensure that the
gasket is clean.
• Patented sealing process individually seals membranes
preventing lateral flow and crosstalk
Step-by-step Combinatorial Screen
AcroPrep Plate
Inside individual wells
Spinner
In multiple
tubes
Incubation/Binding
Directly in plates
(no weeping)
In multiple
tubes
Flow-through
collection
Single collect using
vacuum manifold
Multiple
collections
Washing
Single collect using
vacuum manifold
Multiple
collections
Elution
Single collect using
vacuum manifold
Multiple
collections
• Rigid single-piece construction
• Conforms to ANSI/SBS x. 2004 standards for automation
• Proprietary design minimizes solution/sample weeping
Materials and Methods
Vacuum Filtration
AcroPrep 96 filter plates (Pall Life Sciences)
0.2 µm Bio-Inert® membrane (PN 5042)
0.45 µm GHP membrane (PN 5030)
The use of low biomolecule binding membranes
minimizes the sample losses due to non-specific
binding to the plates. In this study, both filtration media
demonstrate effective bead retention and low non-specific
binding of the proteins.
Mixing Resins
with Samples
Sample Incubation
1. Mix 100 µL of cell lysate with 20 µL of metal ion resin
directly in an AcroPrepTM 96 GHP or Bio-Inert® filter
plate. For experiments shown in Figures 1, 3, 4, 5, 6,
Ni-NTA* (Qiagen) resin was used. TALON* (BD Bioscience)
resin was used in Figure 3. HiTrap Chelating Sepharose
(Amersham Biosciences) was used in Figure 2 (charging
with Metal Ions described below).
2. Incubate at 4 °C with gentle rocking for 30 min.
3. Alternatively, incubation can be performed in microfuge
tubes and transferred into wells of the AcroPrep 96 filter
plate for filtration.
Vacuum Filtration
1. Place the AcroPrep 96 filter plate on the vacuum manifold
and apply vacuum at 25.4 cm Hg (15 in. Hg) for 1 min.
Collect the flow-through in a 96-well collection plate.
2. Wash the resin with 200 µL of washing buffer. Apply
vacuum at 25.4 cm Hg (10 in. Hg) for 1 min. Collect
the wash in fresh 96-well collection plates. Repeat twice.
3. Elute with 100 µL of elution buffer unless otherwise specified.
Apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min. Collect
the elution in fresh 96-well collection plates. Repeat twice.
Charging Chelating Sepharose with Metal Ions For
Custom Resin Screens
1. Pipette 40 µL of 50% HiTrap* Chelating Sepharose
(Amersham Biosciences) slurry into each well of the
AcroPrep 96 filter plate.
Results and Discussion
Sample Incubation Directly in the AcroPrep 96
Filter Plate
Because the AcroPrep 96 filter plates incorporate low
protein-binding filter media and are uniquely designed to
prevent weeping from wells, it is possible to perform sample
incubation directly in the wells of filter plate. To test this
procedure, the sample/resin slurries were incubated either
in a microfuge tube (Figure 1, left panel) or directly in a well
of an AcroPrep 96 filter plate with 0.2 µm Bio-Inert membrane
(Figure 1, right panel) for 30 min. at 4 ºC. The incubation of
sample/resin slurry directly in the wells of an AcroPrep 96
filter plate with 0.2 µm Bio-Inert membrane resulted in similar
binding efficiency as the incubation in the microfuge tubes.
The absence of the His-tagged protein in the flow through
lanes from both panels indicated the complete binding of the
protein to the resin. The recovery of His-tagged protein was
similar for both procedures. In addition, by reducing the
volume of each elution aliquot from 200 µL (left panel) to 50 µL
(right panel), the protein is completely eluted within the first
100 µL (right panel). This result demonstrates that sample
can be incubated in the wells of the AcroPrep 96 filter plates,
simplifying the handling procedure and saving time for
the analyst.
Figure 1.
Samples Can be Incubated Directly in a Filter Plate
Incubation in
Centrifuge Tubes
Incubation in
AcroPrep 96 Plates
M L FT W1 W2 E1 E2 E3
M FT E1 E2 E3
2. Place the AcroPrep 96 plate on the vacuum manifold
and apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min.
3. Wash with 200 µL distilled water. Apply vacuum at
25.4 cm Hg (10 in. Hg) for 1 min. Repeat twice.
4. Mix the resin with 20 µL of the following solution: 0.1M
0.1M NiCl2, 0.1M CoSO4, 0.1M CuSO4 and 0.1M ZnCl2,
respectively. Incubate for 5 min. at room temperature.
5. Apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min.
6. Wash with 200 µL distilled water. Apply vacuum at
25.4 cm Hg (10 in. Hg) for 1 min. Repeat twice.
Aliquots of Ni-NTA resin (Qiagen) were mixed with E. coli inclusion body
lysate containing a His-tagged TEV protease construct (load) as described
in Step-by-step Procedures. The slurry was either incubated in a microfuge
tube and then transferred to a filter plate (left panel) or incubated directly
in a well of an AcroPrep 96 filter plate with 0.2 µm Bio-Inert membrane
(right panel). After washing, the samples were serially eluted with either
3 X 200 µL (left panel) or 3 X 50 µL (right panel) of elution buffer. The load
(L), flow through (FT), wash(W1 and W2), and elution (E1, E2, and E3) were
analyzed by SDS-PAGE. The incubation of sample/resin slurry directly in
the wells of the filter plate gave similar recoveries to those incubated
in microfuge tubes, allowing the simplification of sample handling. Based
on this observation, the on-plate incubation procedure was used for the
subsequent experiments.
Custom Resin Screens
The AcroPrep™ 96 filter plate allows the parallel screening of
metal ions for the purification of a given protein. To select
the suitable metal ion, chelating resins can be manually
charged with different divalent metal ions. The charged
resins are subsequently used in small-scale purification trials
to determine the efficiency of the resin. Figure 2 depicts an
example of such application. Aliquots of Chelating Sepharose
(Amersham Biosciences) resin were charged with Ni2+, Co2+,
Cu2+, and Zn2+, respectively, in the individual wells of an
AcroPrep 96 filter plate with 0.45 µm GHP membrane.
The charged resin was then mixed with E. coli cell lysate (L)
containing a His-tagged human Brf2 truncation construct
(courtesy of Dr. L. Joshua-Tor of CSHL). As shown in Figure 2,
we determined that both Ni2+ and Co2+ charged resins
performed well in purifying the tagged protein while Cu2+
and Zn2+ charged resins should not be used. Based on the
result using AcroPrep 96 filter plates, we have been able to
successfully select the metal ions to be used in purifying the
target protein.
Figure 2.
Screening Manually Charged Resins
Ni2+
Screening Commercially Available Resins
The use of an AcroPrep 96 filter plate allows the parallel
validation of different commercial resins for the purification
of the protein of interest. Reliable purification of human Brf2
truncation construct from the commercially charged resins
confirms the ion selection made in Figure 2. We tested
two commercial resins; one pre-charged by Ni2+, the other
pre-charged by Co2+. Both resins performed well for the
protein tested. Because of the popularity of His-tagged
recombinant protein, multiple choices of pre-charged IMAC
resin are now commercially available for researchers. By using
an AcroPrep 96 filter plate, researchers can conveniently screen
various resins from different sources and select a pre-charged
resin for subsequent purification procedures.
Figure 3.
Use of Pre-charged Resins
TALON (Co2+)
Ni-NTA
M
L
FT
W
E1 E2 E3
FT W
E1 E2 E3
Co2+
M L FT W1 W2 1 E2 E3
M FT W1 W2 E1 E2 E3
Cu2+
M FT W1 W2 E1 E2 E3
Zn2+
M FT W1 W2 E1 E2 E3
Validation of multiple commercial metal-chelating resins can be easily
performed using AcroPrep 96 filter plates. The same lysate samples in
Figure 2 were incubated in the wells of an AcroPrep 96 plate with
GHP membrane with either Ni-NTA* resin (Qiagen) or TALON* resin
(BD Bioscience), respectively. The load (L), flow through (FT), wash (W),
and elution (E1, E2, and E3) were analyzed by SDS-PAGE.
The use of an AcroPrep 96 filter plate can help in the selection of the metal
ions for successful IMAC purification. Aliquots of Chelating Sepharose
(Amersham Biosciences) resin were charged with Ni2+, Co2+, Cu2+, and
Zn2+, respectively, in the individual wells of an AcroPrep 96 filter plate with
0.45 µm GHP membrane. The charged resin was then mixed with E. coli
cell lysate (L) containing His-tagged human Brf2 truncation construct. The
purification was a performed under native condition as described in
Step-by-step Procedure. The flow through (FT), wash (W1 and W2), and
elution (E1, E2, and E3) were analyzed with SDS PAGE.
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Optimizing IMAC Elution Conditions
The use of a multi-well filter plate facilitates the side-by-side
testing of different elution conditions for protein(s) of interest.
This is particularly helpful when a single-step elution condition
is to be used instead of an elution gradient. In an AcroPrep 96
filter plate with 0.45 µm GHP membrane (PN 5030), we tested
three elution conditions in parallel for the purification of the
His-tagged TEV protease. As shown in Figure 4, with all
things being equal, the best recovery of the protein under
denaturing condition was obtained with elution at pH 3.
This result demonstrated that the AcroPrep 96 filter plate
could be used to optimize the elution conditions for IMAC
purification procedures.
Small amount of resin (40 µL) was sufficient for the complete
binding of the His-tagged protein as indicated by the
disappearance of target protein in the flow through (arrows)
as well as the amount of eluted protein reaching plateau.
In both experiments, we have found optimum resin-to-sample
ratio was 1:5 (volume). This information was useful in
calculating the amount of resin to use in the large-scale
purification, minimizing the cost of the sample preparation.
Figure 5.
Titration of Sample Load versus Resin Volume
Variable Sample
Figure 4.
Optimizing Elution
M
L
5 10 20 40
Elution
5 10 20 40
H5
H4
Elu
tio
n
,p
,p
Elu
tio
n
H3
,p
Elu
tio
n
Wa
sh
wth
Flo
ad
Lo
rke
r
rou
gh
Sample Volume
Ma
FT
Variable Resin
M
Resin Volume
L
FT
5 10 20 40
Elution
5 10 20 40
Pall Life Sciences AcroPrep 96 filter plate facilitates the optimization of
elution conditions for IMAC purification. Aliquots of Ni-NTA* resin (Qiagen)
were mixed with E. coli inclusion body lysate (load) containing a His-tagged
TEV protease construct. The purification was performed as described in
Step-by-step Procedures. After washing, the samples were eluted using
three elution buffers with different pH as indicated.
Optimizing Resin and Sample Loads
The AcroPrep 96 filter plate with 0.45 µm GHP membrane
(PN 5030) was used to determine the loading capacity of the
selected IMAC resin prior to large-scale sample purification.
In a single experiment, researchers can perform multiple
titrations of resin volume and sample load (Figure 5). For a
given resin volume of 20 µL, increasing amount of the sample
was loaded (upper panel). The resin was saturated at 100 µL
sample load as indicated by the appearance of target protein
in the flow through (arrows) when larger volume is loaded.
Meanwhile, for a given sample volume of 200 µL, increasing
amount of resin was used for the purification (lower panel).
AcroPrep 96 filter plates can help optimize the amount of resin to be used in
an IMAC application. Aliquots of Ni-NTA* resin (20 µL, Qiagen) were mixed
with different volumes (5, 10, 20, and 40 µL) of E. coli inclusion body lysate (L)
containing a His-tagged TEV protease construct (top panel). In addition, four
different volumes of Ni-NTA resin (5, 10, 20, and 40 µL, Qiagen) were mixed
with 200 µL of the same lysate (bottom panel). The flow through (FT) and
elution were analyzed by SDS PAGE gel.
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Well-to-well Reliability using Multi-well Minicolumns
High throughput sample processing in proteomic research
requires the protein recovery be consistent from well to well
in the filter plates. As shown in Figure 6, the elution from
96 identical samples processed by minicolumns in a single
AcroPrep filter plate with 0.45 µm GHP membrane (PN 5030)
was consistent from well to well as judged by the intensity
of protein bands in SDS PAGE gels. In addition, the protein
concentration of each eluted sample was quantified by
BCA assay, giving a CV of 9.3%. This result indicated
superb well-to-well reliability of the AcroPrep 96 filter plate
in processing multiple samples.
Figure 6.
Well-to-well Reproducibility
Conclusions
IMAC purification is widely used for sample preparations
in current proteomic studies. We have demonstrated in
this report the use of multi-well filter plates to perform high
throughput IMAC. The low-protein binding and no-weeping
properties of the AcroPrep 96 filter plate are ideal for
convenient incubation of the sample directly in wells.
The biomolecule-friendly AcroPrep 96 filter plate allows the
researcher to rapidly and reliably screen numerous samples
under a variety of conditions to determine protein purification
parameters. A single filter plate can be matrixed to:
• Screen for metal ions for both custom and
pre-charged resins
• Optimize elution conditions
• Optimize resin-to-load ratio
In addition, Pall Life Sciences’ AcroPrep 96 filter plate
shows consistent well-to-well performance, giving protein
biochemists an edge in the development of protein
purification protocols.
References
1. Porath J., Carlsson J., Olsson I., and Belfrage G. (1975)
Metal chelate affinity chromatography, a new approach to
protein fractionation. Nature 258, 598-599.
2. Hochuli E., Bannwarth W., Dobeli H., Gentz R., and Stuber
D. (1988) Genetic approach to facilitate purification of
recombinant proteins with a novel metal chelate adsorbent.
Biotechnology 6: 1321-1325.
Ninety-six 20 µL aliquots of Ni-NTA* resin (Qiagen) were mixed with
E. coli inclusion body lysate containing a His-tagged TEV protease
construct. The purification was performed as described in Step-by-step
Procedures. The eluted samples from each well were analyzed using SDS
PAGE. The protein concentration of the elution from separate wells was
measured by BCA assay. The coefficient of variation was 9.3%, indicating
excellent well-to-well consistency.
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3. Desalting/Buffer Exchange for Biomolecules Using
AcroPrep 96 Ultrafiltration Filter Plates, Pall Life Sciences
PN33309.
4. Lysate Clearance for Prokaryotic DNA Isolation Using the
AcroPrep 96 Filter Plate, Pall Life Sciences PN33308.
5. Automated Purification of Combinatorial Libraries
Using AcroPrep 96 Filter Plate with GHP Membrane,
Pall Life Sciences PN33245.
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10/04, 1.5k, GN04.0943
PN33354