Ni-NTA Agarose - Biontex Laboratories

Ni-NTA Agarose
For purification of histidine-tagged fusion proteins
For ordering information, MSDS, publications and application notes see www.biontex.com
Description Cat. No. Size
Ni-NTA Agarose R040-01.1 1.0 ml resin
Ni-NTA Agarose R040-05.1 5.0 ml resin
Ni-NTA Agarose R040-10.1 10.0 ml resin
Ni-NTA Agarose R040-50.1 50.0 ml resin
Ni-NTA Agarose R040-50.2 100.0 ml resin (2 × 50 ml)
Volumes correspond to settled agarose resin.
Shipping:
At room temperature
Storage:
2 - 8 °C; do not freeze
Stability:
Best before: see label
Use:
Only for research purposes in vitro, not intended for human or animal
diagnostic, therapeutic or other clinical uses.
Description
Biontex Ni-NTA Agarose provides a simple and very effective means of purifying recombinant
proteins fused with a 6-fold histidine tag (His-tag). The His-tag does not usually affect the
bioactivity of the protein in question, and the purification procedure is simple.
The agarose consists of the tridentate chelating agent, nitrilotriacetic acid (NTA), covalently
coupled to 6 % cross-linked agarose beads, and is loaded with Ni 2+ ions. Immobilized Nickel
selectively binds exposed histidine residues and allows recombinant His-tagged proteins to be
purified directly from crude bacterial lysates under either native or denaturing conditions. Resin
allows batch or column purifications.
Ni-NTA Agarose Manual EN 201312121403 1

Content
Description................................................................................................................................. 1
Content ..................................................................................................................................... 2
1. General Information..................................................................................................................... 3
1.1 Specifications............................................................................................................................... 3
1.2 Flow Rates................................................................................................................................... 3
1.3 Explanatory Remarks .................................................................................................................... 3
1.4 Native or Denaturing Conditions ..................................................................................................... 3
1.5 Proposal of Sample Preparation under Native Conditions ................................................................... 4
Example of Native Buffer Compositions:......................................................................................... 5
1.6 Proposal of Sample Preparation under Denaturing Conditions............................................................. 5
Isolation of Inclusion Bodies ......................................................................................................... 5
Solubilization of Inclusion Bodies................................................................................................... 5
Usage of Denaturing Buffers in Working Instructions ....................................................................... 6
2. Working Instructions: Batch Purification ..................................................................................... 7
2.1 Biontex Ni-NTA Agarose-Preparation ............................................................................................... 7
a) Elimination of the Preservative............................................................................................... 7
b) Equilibration of the Resin ...................................................................................................... 7
2.2 Sample Application ....................................................................................................................... 8
2.3 Washing ...................................................................................................................................... 8
2.4 Elution ........................................................................................................................................ 8
2.5 Regeneration and Storage ............................................................................................................. 9
3. Working Instructions: Gravity Purification................................................................................... 9
3.1 Biontex Ni-NTA Agarose-Preparation ............................................................................................... 9
3.1.1. Elimination of the Preservative ....................................................................................... 9
3.1.2. Equilibration of the Pre-Packed Column ......................................................................... 10
3.2 Sample Application ..................................................................................................................... 10
3.3 Washing .................................................................................................................................... 11
3.4 Elution ...................................................................................................................................... 11
3.5 Regeneration and Storage ........................................................................................................... 11
4. Working Instructions: Spin Purification ..................................................................................... 12
4.1 Biontex Ni-NTA Agarose Preparation ............................................................................................. 12
4.1.1. Elimination of the Preservative ..................................................................................... 12
4.1.2. Equilibration of the Pre-Packed Spin Column .................................................................. 13
4.2 Sample Application ..................................................................................................................... 13
4.3 Washing .................................................................................................................................... 13
4.4 Elution ...................................................................................................................................... 13
4.5 Regeneration and Storage ........................................................................................................... 14
5. Working Instructions: FPLC TM Purification ................................................................................. 14
5.1 Column Packaging, Elimination of the Preservative and Equilibration of the Resin ............................... 14
5.2 Sample Application ..................................................................................................................... 15
5.3 Washing .................................................................................................................................... 15
5.4 Elution ...................................................................................................................................... 15
5.5 Regeneration and Storage ........................................................................................................... 16
6. Regeneration and Storage.......................................................................................................... 16
Regeneration Protocol 1 (Decontamination):................................................................................. 16
Regeneration Protocol 2 (Removal of Metal Ions): ......................................................................... 16
7. Miscellaneous............................................................................................................................. 17
7.1 Compatibility of Reagents ............................................................................................................ 17
7.2 Tips .......................................................................................................................................... 18
7.3 Important Information ................................................................................................................ 18
7.4 Warranty ................................................................................................................................... 18
8. Related Products........................................................................................................................ 19
2 Ni-NTA Agarose Manual EN 201312121403

1. General Information
1.1 Specifications
Applications
Binding / Loading capacity
Bead structure
Affinity chromatography of 6×histidine-tagged fusion proteins in
batch or gravity flow processes
>50 mg of 6×histidine-tagged fusion protein per 1 ml agarose
Cross-linked 6 % agarose
Bead size Spherical, standard ca. 50 – 170 µm
Max. pressure
Formulation
Antimicrobial agent
Shelf life
2.6 psi (0.18 bar)
Unbuffered suspension in 30% ethanol
30 % ethanol
5 years (after date of certificate of analysis)
Storage 2 – 8 °C
1.2 Flow Rates
Recommended standard-flow rate
Maximum linear flow rate
1 ml / min
300 cm / h
1.3 Explanatory Remarks
Separations can be performed by batch purification, gravity flow and spin columns, or by columns
attached to a low-pressure pump such as a peristaltic pump or FPLC TM -System.
Elution is possible by several techniques, including lowering the pH, or by using a metal complexing
agent such as imidazole or EDTA in the buffer. There is a balance between the concentration of
imidazole required to elute the His-tagged protein, and the amount needed to avoid nonspecific
binding of contaminants. Generally a higher imidazole concentration in the equilibration buffer will
give a higher purity of protein, although it will compromise the yield.
Column chromatography may be monitored at 280 nm to obtain a profile of the elution peak, thus
enabling dilution of the eluate to be avoided.
It is recommended that an aliquot of each step is retained for SDS-PAGE analysis. This will assist in
optimizing the purification. It is also a means of monitoring the procedure to ensure that it has
worked.
1.4 Native or Denaturing Conditions
Biontex Ni-NTA Agarose may be used under denaturing or native conditions.
It is important to determine whether the His-tagged protein is soluble in the cytoplasm, or is
present in inclusion bodies. If the latter, the protein must be solubilized with denaturants such as
urea of guanidine hydrochloride before purification. Denaturants ensure that otherwise insoluble
His-tagged proteins can be purified, and will cause a conformational change that exposes the Histag,
enabling more efficient binding to the Ni-NTA Agarose. Denaturing conditions, however,
generally result in the complete loss of biological and biochemical activity of the proteins being
purified. It is preferable to lyse the cells in the milder denaturant, urea, so that the cell lysate can
be analyzed directly on a SDS polyacrylamide gel. GuHCl is a more efficient solubilization and cell
lysis reagent, however, and may be required to solubilize some proteins. Since fractions which
contain GuHCl will precipitate with SDS when loaded onto an SDS polyacrylamide gel, they must
either be diluted (1:6), dialyzed before analysis, or separated from GuHCl by TCA precipitation.
Ni-NTA Agarose Manual EN 201312121403 3

Some membrane proteins may not be solubilized under chaotropic conditions, but may be able to
be solubilized using detergents.
Purification under native conditions allows the co-purification of associated proteins, and allows
further study of biological or biochemical activity such as enzyme activity, antibody binding or
receptor activity.
If purification is to be performed under native conditions, His-tagged protein must be soluble under
these conditions. However, even if the majority of His-tagged protein is present in insoluble
inclusion bodies, some soluble protein may still be present and can be purified under native
conditions.
Purification under native conditions may result in co-purification of unrelated, untagged proteins
than when denaturing conditions are employed. Nonspecific binding is minimized by adding a low
concentration of imidazole to the native lysis buffer and native equilibration buffer.
Note: Buffers for native conditions differ from buffers for denaturing conditions!
1.5 Proposal of Sample Preparation under Native Conditions
1. Collect the cells from the 'overnight' culture by centrifuging (7,000 – 8,000 x g for 10 min or
4,000 x g for 15 min or 1,000 – 1,500 x g for 30 min at 4°C). The volume of culture used
depends on the expression level of the desired protein. Freeze the pellet for further use.
2. Thaw the cell pellet on ice for approx. 15 minutes.
3. Cell lysis methods: Cell lysis can be performed using various methods
a) Mechanical, such as sonication, homogenization, grinding or pressurized decomposition
using the French-press principle. For these, resuspend the pellet cells in cold native
resuspension buffer, using 5 volume units of resuspension buffer for each volume unit of
wet pelleted cells. If using sonication or homogenization, minimize the treatment time as far
as possible, use ice to cool the suspension and pulse in short bursts - typically 5 to 6 10-
second pulses interspersed by 5-second breaks. Excessive ultrasound or homogenization
treatment may destroy protein functions. Avoid freezing samples, as this may cause
denaturization of the fusion protein and result in co-purification with the original protein.
b) Cells should preferably be lysed chemically in native lysis buffer. To do this, suspend the
pelleted cells in the buffer (2 - 5 ml lysis buffer per 1 g wet pelleted cells) on ice for approx.
30 minutes.
The lysis buffer contains 10 mM imidazole to minimize binding of untagged proteins
(contaminants) and optimize purification while reducing the number of washing steps.
If the fusion protein fails to bind under these conditions reduce the concentration of
imidazole to 1 – 5 mM.
The maximum concentration of imidazole (for strongly binding proteins) is 20 mM.
4. Sonicate the suspension (approx. 10 x at 200-300 W, with 10s cooling interval between each
sonication).
5. If the lysate is still viscous add 5 mg/ml DNase I (or RNase I (5 µg/ml) and RNase A (10 µg/ml)
of preferred) and incubate on ice for 10 - 15 min.
6. Centrifuge the crude lysate at 10,000 - 12,000 x g for 15 - 30 min at 4ºC and remove the
supernatant, leaving all cell debris behind. Retain a specimen of lysate (approx. 5 µl) for SDS-
PAGE analysis.
7. Run an SDS-PAGE analysis to determine the protein concentration.
4 Ni-NTA Agarose Manual EN 201312121403

Example of Native Buffer Compositions:
Native lysis buffer for chemical extraction only, must be freshly prepared
50 mM NaH 2 PO 4 pH 8.0 (over NaOH) 10 mM Imidazole
300 mM NaCl 1.0 mg/ml Lysozyme
Native resuspension buffer (for mechanical extraction)
50 mM NaH 2 PO 4 pH 8.0 (over NaOH) 10 mM Imidazole
300 mM NaCl
1.6 Proposal of Sample Preparation under Denaturing
Conditions
Cells may be lysed in either 6 M GuHCl No. 1) or 8 M urea (No. 2) denaturing lysis buffers. Urea is
a milder denaturant, and the lysate can be analyzed directly by SDS-PAGE. GuHCl is a stronger
denaturant and may be necessary to solubilize some proteins. Where GuHCl is used it will be
necessary to either dilute (1:6) with water, or dialyze before SDS-PAGE analysis.
Isolation of Inclusion Bodies
1. Thaw the cell pellet from an E.coli expression culture on ice (if frozen).
2. Resuspend 1 g of pelleted, wet cells in 5 ml buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM
imidazole, pH 8.0) on ice.
3. Pipette up and down or stir until complete resuspension is reached without visible cell
aggregates.
4. Add lysozyme to a final concentration of 1mg/ml.
5. Stir the solution on ice for 30 min.
6. Sonicate the suspension on ice according to the instructions and check the appearance of the
sample after sonication. If the lysate is still viscous add 5 mg/ml DNase I and stir on ice for 15
min.
7. Centrifuge the crude lysate at 10,000 x g for 30 min at 4ºC to collect the inclusion bodies.
8. Discard supernatant and keep pellet on ice.
Solubilization of Inclusion Bodies
1. Resuspend the pellet in 10 ml 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0.
2. Centrifuge at 10,000 x g (30 min at 4ºC) and discard supernatant.
3. Add 2.0 ml (per g wet cells) 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, 8 M urea pH 8.0.
4. Homogenization or sonication may be necessary to resuspend the pellet.
5. Dissolve the inclusion bodies by stirring on ice for 60 min.
6. Centrifuge at 10,000 x g for 30 min (at 20 ºC) to eliminate insoluble material and transfer the
supernatant to a clean tube.
7. Centrifuge until the supernatant is clear and save it.
Resuspension buffer
50 mM NaH 2 PO 4 pH 8.0 (over NaOH) 300 mM NaCl
10 mM Imidazole
Lysis buffer (urea)
Lysis buffer (GuHCl)
50 mM NaH 2 PO 4 pH 8.0 (over NaOH) 50 mM NaH 2 PO 4 pH 8.0 (over NaOH)
300 mM NaCl 300 mM NaCl
10 mM Imidazole 10 mM Imidazole
8 M Urea 6 M GuHCl
Ni-NTA Agarose Manual EN 201312121403 5

Usage of Denaturing Buffers in Working Instructions
The procedure is similar to purification under native conditions except that the sample and the
buffers loaded on the column contain 8 M urea (or 6M GuHCl):
Binding buffer (urea)
Binding buffer (GuHCl)
50 mM NaH 2 PO 4 pH 8,0 (over NaOH) 50 mM NaH 2 PO 4 pH 8,0 (over NaOH)
10 mM Imidazole 10 mM Imidazole
300 mM NaCl 300 mM NaCl
8 M Urea 6 M GuHCl
Washing buffer (urea)
Washing buffer (GuHCl)
50 mM NaH 2 PO 4 pH 8.0 (over NaOH) 50 mM NaH 2 PO 4 pH 8.0 (over NaOH)
20 mM Imidazole 20 mM Imidazole
300 mM NaCl 300 mM NaCl
8 M urea 6 M GuHCl
Elution buffer (urea)
Elution buffer (GuHCl)
50 mM NaH 2 PO 4 pH 8.0 (over NaOH) 50 mM NaH 2 PO 4 pH 8.0 (over NaOH)
250 mM Imidazole 250 mM Imidazole
300 mM NaCl 300 mM NaCl
8 M Urea 6 M GuHCl
Note: Due to the dissociation of urea, the pH of denaturing buffers should be adjusted
immediately prior to use. Do not autoclave.
6 Ni-NTA Agarose Manual EN 201312121403

2. Working Instructions: Batch Purification
The following summarized procedure is adapted for the purification of His-tagged protein under
native conditions.
The recombinant proteins often form insoluble inclusion bodies. If this occurs the inclusion bodies
need to be rendered soluble by purifying under denaturing conditions, using, for example, urea or
guanidine chloride at relevant stages (see Chapter 1.4).
2.1 Biontex Ni-NTA Agarose-Preparation
The volume of Biontex Ni-NTA Agarose required is determined by the amount and nature of the
His-tagged protein to be purified.
a) Elimination of the Preservative
Determine the quantity of Biontex Nickel NTA Agarose
needed for your purification, following the
recommendations below.
1 ml gel corresponds to 2 ml of
50 % (v/v) Biontex Ni-NTA
Agarose suspension.
Note: Binding capacity will vary for each HIS-tagged protein. The yield of HIS-tagged
protein depends on various parameters such as nature of the fusion protein,
expression host, culture conditions, etc. Biontex Ni-NTA Agarose has a binding
capacity benchmark of > 50 mg of HIS tagged protein / ml gel.
Note: Binding capacity can be affected by several factors such as sample
concentration, binding buffer, or flow rate during sample application.
Option A: Gently shake the bottle of Nickel NTA Agarose to achieve a homogeneous suspension.
Immediately pipette the suspension (2 ml of Biontex Ni-NTA Agarose suspension per ml of gel
volume required) into an appropriate tube. Sediment the gel by centrifuging at 500 x g for 5
minutes, carefully decant the supernatant and discard it.
Option B: Manually shake the bottle of resin to obtain a homogeneous suspension of beads and
preservative. Invert the bottle of resin several times, filter the resin and place in a container.
b) Equilibration of the Resin
Add 10 bed volumes of binding buffer to equilibrate the gel by mixing thoroughly to achieve a
homogeneous suspension. Sediment the gel by centrifuging at 500 x g for 5 minutes, carefully
decant the supernatant and discard it.
Ni-NTA Agarose Manual EN 201312121403 7

Note: Binding buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0.
Binding buffer: The choice of buffer depends on the specific properties of the protein. The buffer
used most frequently is phosphate (50 mM is recommended). The pH of binding buffers generally
leads to neutrality (7.0 – 8.0).
Note: In some cases it is necessary to increase the concentration of imidazole (1 –
20 mM) to increase the selectivity of the binding of the target protein. It is
important to use high purity imidazole to avoid affecting the O.D. 280 nm and the
presence of agents like EDTA or citrate at all times.
2.2 Sample Application
Once the resin is equilibrated, the sample containing the fused protein for purification is applied
(see Chapter 1.5 und 1.6).
Mix the suspension gently for 30 - 60 min at room temperature.
In some cases binding may be facilitated by slightly increasing contact time.
Centrifuge the suspension at 500 x g for 5 minutes to sediment the resin. Carefully decant the
supernatant and discard it.
2.3 Washing
Note: SDS-PAGE or O.D. 280 nm analyses will give a good indication of how much
washing is needed.
Note: Washing buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0.
1. Wash the gel by adding 10 bed volumes of wash buffer.
2. Invert to mix, then centrifuge the suspension at 500 x g for 5 minutes to sediment the resin.
3. Carefully decant the supernatant and discard it.
4. Repeat the washing step twice (total wash 3 x 10 bed volumes of binding buffer).
2.4 Elution
Add 1 bed volume of elution buffer to the gel. Mix thoroughly for 10 min at room temperature.
Sediment the gel by centrifuging at 500 x g for 5 minutes, carefully decant or pipette the
supernatant into a new tube and store on ice. Repeat the elution step twice or more and pool the
fractions containing the purified protein.
Note: Elution buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole pH 8.0.
A significant amount of His-tagged protein may remain bound to the resin. Conditions (volumes,
times, temperatures) used for elution may vary among His-tagged proteins. Eluates should be
8 Ni-NTA Agarose Manual EN 201312121403

monitored (Bradford protein assay, SDS-PAGE or measurement of absorbance at 280 nm to
determine the yield of the eluted His-fused protein).
For proper storage removal of the imidazole by ultrafiltration, dialysis, or precipitation with
ammonium sulphate is recommended.
For most of the applications it is not necessary to eliminate the His-tag. However, this elimination
is necessary for certain applications such as X-ray crystallography or RMN, where the protein
structure is to be determined later. For these purposes, a His-tag is usually spliced to the protein at
a protease cleavage site.
2.5 Regeneration and Storage
See Chapter 6
3. Working Instructions: Gravity Purification
The following summarized procedure is adapted for the purification of His-tagged protein under
native conditions.
The recombinant proteins often form insoluble inclusion bodies. If this occurs the inclusion bodies
need to be rendered soluble by purifying under denaturing conditions, using, for example, urea or
guanidine chloride at relevant stages (see Chapter 1.4).
We recommend using Biontex Empty Columns depending on total volume size (Empty L-Columns
(Cat. Nº. P-50L) with capacity of 12 ml or Empty XL-Columns (Cat. Nº. P-50XL) with capacity of
35 ml).
Note: All solvents and product solutions may be degassed before added to a column.
3.1 Biontex Ni-NTA Agarose-Preparation
3.1.1. Elimination of the Preservative
The volume of Biontex Ni-NTA Agarose required is determined by the amount and nature of the
His-tagged protein to be purified.
Note: 100 ml of the original 50%
suspension corresponds to 50 ml
of gel.
Note: Binding capacity will vary for each HIS-tagged protein. The yield of HIS-tagged
protein depends on various parameters such as the nature of the fusion protein,
expression host, culture conditions, etc. Biontex Ni-NTA Agarose has a binding
capacity benchmark of > 50 mg of HIS tagged protein / ml gel.
Note: Binding capacity can be affected by several factors such as sample
concentration, binding buffer or flow rate during sample application.
Ni-NTA Agarose Manual EN 201312121403 9

1. Determine the quantity of Nickel NTA Agarose needed for your purification.
2. Gently shake the bottle of Nickel NTA Agarose to achieve a homogeneous
suspension. Immediately pipette sufficient suspension into an appropriate
empty column.
3. Remove first the upper and then the lower cap of the column, to allow
elimination of the preservative by gravity flow.
3.1.2. Equilibration of the Pre-Packed Column
1. Equilibrate the column with 5 bed volumes of binding buffer. Add the
binding buffer to the upper part of the column and ensure no air has
been trapped.
2. Mix manually by inverting the pre-packed column and discard the
supernatant.
3. Repeat the equilibration step twice.
Note: Binding buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0.
Binding buffer: The choice of buffer depends on the specific properties of the protein. The buffer
used most frequently is phosphate (50 mM is recommended). The pH of binding buffers generally
leads to neutrality (7.0 – 8.0).
Note: In some cases it is necessary to vary the concentration of imidazole (1 –
20 mM) to increase the selectivity of the binding of the target protein. It is
important to use high purity imidazole to avoid affecting the O.D. 280 nm. It is
important to avoid the presence of agents like EDTA or citrate at all times.
3.2 Sample Application
1. Add the sample containing the His-tagged protein to be purified through the top of the column,
keeping the sample and resin in contact 30 – 60 minutes before removing the bottom cap.
In some cases a slight increase of contact time may facilitate binding.
2. Mix manually by inverting the pre-packed column. Remove the lower cap of the column and
discard the supernatant.
10 Ni-NTA Agarose Manual EN 201312121403

3.3 Washing
Note: SDS-PAGE or O.D. 280 nm analysis will give a good indication of how much
washing is needed.
Note: Washing buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0.
1. Close the column outlet using the cap.
2. Add the binding buffer (10 bed volumes) through the top to eliminate all proteins that have not been
retained in the column.
3. Close the column inlet using the cap and mix manually by inverting the pre-packed column.
4. Remove the lower cap of the column and discard the supernatant.
5. Repeat the washing step twice (total wash 3 x 10 bed volumes of washing buffer).
6. Retain aliquots (5 µl) for SDS-PAGE or O.D. 280 nm analysis.
3.4 Elution
Note: Elution buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM
imidazole pH 8.0.
1. Close the column outlet using the cap.
2. Add 1 bed volume of elution buffer to the column.
3. Close the column inlet using the cap and mix thoroughly for 10 min at
room temperature.
4. Sediment the gel, remove the end cap, collect the eluate in a new tube and store on ice.
5. Repeat the elution step twice and pool the collected eluates.
6. Retain aliquots (5 µl) for SDS-PAGE or O.D. 280 nm analysis.
It is possible that a significant amount of His-tagged protein may remain bound to the resin.
Conditions (volumes, times, temperatures) used for elution may vary among His-tagged proteins.
Eluates should be monitored (Bradford protein assay, SDS-PAGE or measurement of the
absorbance at 280 nm to determine the yield of the eluted His-fused protein).
For proper storage removal of the imidazole by ultrafiltration or dialysis is recommended.
For most of the applications it is not necessary to eliminate the His-tag. However, this elimination
is necessary for certain applications such as X-ray crystallography or RMN, where the protein
structure is to be determined later. For these purposes, a His-tag is usually spliced to the protein,
at a protease cleavage site.
3.5 Regeneration and Storage
See chapter 6
Ni-NTA Agarose Manual EN 201312121403 11

4. Working Instructions: Spin Purification
The following summarized procedure is adapted for the purification of His-tagged protein under
native conditions. The recombinant proteins often form insoluble inclusion bodies. If this occurs
these need to be rendered soluble by purifying under denaturing conditions, using, for example,
urea or guanidine chloride at relevant stages (see Chapter 1.4).
Note: All solvents and product solutions may be degassed before added to a column.
The following summarized procedure is adapted to the purification of His-tagged protein in spin
columns. We recommend using Biontex Empty Spin Columns (Cat. Nº. SP-50, Capacity 800 µl).
This protocol requires
Empty Spin Columns with
inserted frits of 10 - 20
mm pore size
4.1 Biontex Ni-NTA Agarose Preparation
4.1.1. Elimination of the Preservative
The volume of Biontex Ni-NTA Agarose required is
determined by the amount and nature of the His-tagged
protein to be purified.
Note: 100 ml of the original 50%
suspension corresponds to 50 ml
of gel.
Note: Binding capacity will vary for each HIS-tagged protein. The yield of HIS-tagged
protein depends on various parameters such as the nature of the fusion protein,
expression host, culture conditions, etc. Biontex Ni-NTA Agarose has a binding
capacity benchmark of > 50 mg of HIS tagged protein / ml gel.
Note: Binding capacity can be affected by several factors such as sample
concentration, binding buffer or flow rate during sample application.
1. Determine the quantity of Nickel NTA Agarose needed for purification.
2. Gently shake the bottle of Nickel NTA Agarose to achieve a homogeneous suspension.
3. Immediately pipette 100 µl of the original suspension into an appropriate empty spin column.
4. Remove the lower outlet cap and place the spin column in a collecting tube.
5. Centrifuge at 500 x g for 30 seconds.
12 Ni-NTA Agarose Manual EN 201312121403

4.1.2. Equilibration of the Pre-Packed Spin Column
Note: Binding buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0.
1. Equilibrate the spin column with 500 µl of binding buffer.
2. Mix manually, centrifuge at 500 x g for 30 seconds and discard flowthrough.
4.2 Sample Application
1. Close the spin column outlet using the cap.
2. Add the sample containing the His-tagged protein to be purified through the top of the spin
column, keeping sample and resin in contact for 30 - 60 minutes before removing the bottom
cap.
In some cases a slight increase of contact time may facilitate binding. Binding
capacity can be affected by several factors such as sample concentration.
3. Mix manually by inverting the spin column.
4. Remove the bottom cap and place the spin column in a collecting tube.
5. Centrifuge at 500 x g for 30 seconds and discard the flowthrough.
4.3 Washing
Note: Washing buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0.
Note: Wash the column with binding buffer until the O.D. 280 nm of the eluent
reaches the baseline level.
1. Add 500 µl washing buffer through the top to eliminate all the proteins that have not been
retained in the column.
2. Mix manually by inverting the spin column.
3. Centrifuge at 500 x g for 30 seconds.
4. Discard flowthrough.
5. Repeat the washing step twice.
6. Discard flowthrough between washing steps.
4.4 Elution
Note: Elution buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole pH 8.0.
1. Close the spin column outlet using the cap.
2. Add 500 µl of elution buffer and close the lid.
3. Mix thoroughly for 10 min at room temperature.
4. Centrifuge the gel, remove the end cap, collect the eluate in a new tube and store on ice.
Ni-NTA Agarose Manual EN 201312121403 13

5. Repeat the elution step twice.
6. Pool the collected eluates.
A significant amount of His tagged protein may remain bound to the resin. Conditions (volumes,
times, temperatures) used for elution may vary among His-tagged proteins. Eluates should be
monitored (Bradford protein assay, SDS-PAGE or measurement of the absorbance at 280 nm to
determine the yield of the eluted His-fused protein).
For proper storage removal of the imidazole by ultrafiltration or dialysis is recommended.
For most of the applications it is not necessary to eliminate the His-tag. However, this elimination
is necessary for certain applications such as X-ray crystallography or RMN, where the protein
structure is to be determined later.
For these purposes, a His-tag is usually spliced to the protein, at a protease cleavage site.
4.5 Regeneration and Storage
See chapter 6
5. Working Instructions: FPLC TM Purification
Biontex Ni-NTA Agarose Resin is compatible with common low pressure chromatography columns
and FPLC TM applications. We recommend columns equipped with an adjustable plunger/flow
adapter. Use low rates for the loading step to allow maximal binding of the His-tagged protein. The
flow rate for equilibration, washing and elution can be increased to reduce the purification time
(see Table 1.2).
The following summarized procedure is adapted for the purification of His-tagged protein under
native conditions. The recombinant proteins often form insoluble inclusion bodies. If this occurs the
inclusion bodies need to be rendered soluble by purifying under denaturing conditions, using, for
example, urea or guanidine chloride at relevant stages (see Chapter 1.4).
Note: It is advisable to degas all the solutions before adding to the column to avoid
the formation of bubbles.
5.1 Column Packaging, Elimination of the Preservative and
Equilibration of the Resin
1. Manually shake the bottle to obtain a homogeneous suspension of Biontex Ni-NTA Agarose
Beads/preservative.
2. Place a funnel in the top of the column and slowly run the suspension down the walls of the
column.
Note: It is advisable to add the suspension slowly to avoid the formation of bubbles.
The product may also be degassed before adding to the column.
Note: Make sure no air is trapped under the net.
3. Decant the product and discard most of the residual liquid, leaving 1 cm above the column head
to prevent drying out. This is done either by passing it through the column, or pipetting it from
the top of the column.
4. Repeat previous steps until the desired column height is obtained.
14 Ni-NTA Agarose Manual EN 201312121403

5. Insert the adapter gently into the column head until it begins to displace the liquid.
6. Add distilled water to the purification stream until a constant height (corresponding to the
height of the column) is achieved.
Note: If the desired height is not achieved, repeat steps 1. - 6.
7. When a constant height has been obtained, maintain the flow by adding 5 volumes of distilled
water to completely eliminate the preservative.
8. Equilibrate the column with at least 5 column volumes of binding buffer until the baseline at
280 nm is stable.
Note: Binding buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0.
5.2 Sample Application
Once the resin is equilibrated, the centrifuged or filtered sample is added. In some cases a slight
increase of contact time may facilitate binding. Therefore use low rates for the loading step to allow
maximal binding of the His-tagged protein.
Binding capacity will vary for each His-tagged protein. The yield of His-tagged
protein depends on various parameters such as amino acid composition, 3-D
structure, molecular weight, etc. Nickel NTA Agarose has an orientative binding
capacity of > 50 mg/ml gel (6 x His –GFPuv, ~32 kDa)l
Binding capacity can be affected by several factors such as sample concentration or
the flow rate during sample application. Collect flowthrough and verify that fused
protein has bound.
5.3 Washing
The sample must be washed with the washing buffer (10 – 20 bed volumes) or until the O.D.
280 nm reaches the baseline level.
Note: Washing buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0.
5.4 Elution
Elute the His-tagged protein with 5-10 bed volumes of elution buffer and collect the eluates on ice.
Note: It is important to identify the eluates that contain a majority of pure protein
(by means of SDS-PAGE & Bradford protein assay).
Note: Elution buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8.0.
For proper storage removal of the imidazole by ultrafiltration or dialysis is recommended.
Ni-NTA Agarose Manual EN 201312121403 15

For most of the applications it is not necessary to eliminate the His-tag. However, this elimination
is necessary for certain applications such as X-ray crystallography or RMN, where the protein
structure is to be determined later.
For these purposes, a His-tag is usually spliced to the protein, at a protease cleavage site.
5.5 Regeneration and Storage
See chapter 6
6. Regeneration and Storage
The resin may lose binding points during its life because some protein is retained. Hence, loss of
binding capacity may be observed in successive cycles. Regeneration may be necessary to return
the resin to its initial state. Regeneration involves complete elimination of the retained protein.
In general, column regeneration is always necessary when changing proteins. When continuing
with the same protein, regeneration is recommended when an appreciable diminution in the yield is
observed. The frequency of these stages varies with the protein and the conditions used.
Regeneration Protocol 1 (Decontamination):
1. Solubilize and desorb contaminants by washing the resin with 0.5 M NaOH (30 min).
2. Remove the NaOH solution by washing with 10 bed volumes of distilled water.
3. If you are using the resin directly, wash with 10 bed volumes of 50 mM NaH 2 PO 4 , 300 mM NaCl,
10 mM imidazole, pH 8.0.
4. For storage wash with 2 volumes of 20 % ethanol, resuspend the resin in 20 % ethanol and
store at 2 – 8 ºC.
In some cases the above procedure may not be sufficient depending on the nature of the sample
(for example, if the resin changes colours due to loss or reduction of nickel ions). Regeneration
involves complete elimination of the metal:
Regeneration Protocol 2 (Removal of Metal Ions):
• Wash the resin with 10 bed volumes of distilled water.
• Eliminate the metal from the resin by washing with 10 bed volumes of 100 mM EDTA, pH 8.0.
• Eliminate the excess EDTA: eliminate the residual EDTA before reloading the resin with the
metal by washing the column with 10 bed volumes of distilled water.
• Load the resin with the corresponding metal: once the excess EDTA has been eliminated, add 2
bed volumes of 100 mM metal ion aqueous solution (normally chlorides or sulphates are used).
• Eliminate excess metal: wash with 10 bed volumes of distilled water.
• Prepare usable resin: add 10 volumes of the binding buffer (50 mM NaH 2 PO 4 , 300 mM NaCl,
10 mM imidazole, pH 8.0).
• For storage, wash with 2 volumes of 20 % ethanol, resuspend the resin in 30 % ethanol and
store at 2 – 8 ºC.
16 Ni-NTA Agarose Manual EN 201312121403

7. Miscellaneous
7.1 Compatibility of Reagents
STUDIES REAGENTS COMMENTS
BUFFERS:
DENATURING AGENTS:
ADDITIVES:
DETERGENTS:
• Sodium phosphate
•
• TRIS, HEPES, MOPS
• Sodium chloride
• Urea
• Guanidine-HCl
• Imidazole
• Glycerol
• EDTA
• Ethanol
Non-ionic detergents
(Tween, Triton, Chaps etc.)
• 50 mM pH 8.0 Sodium phosphate
buffer is recommended.
• Coordinate with metal ions,
causing a decrease in binding
capacity. Up to 100mM may be
used.
• Avoids unspecific binding (ionic
interactions).
• At least 0.3M should be used. Up
to 2 M can be used
• Solubilizes protein. Use 8 M for
purification under denaturing
conditions.
• Solubilizes protein. Up to 6 M can
be used.
• Competes with the His-tagged
protein.
• Reduce non specific binding (20
mM)
• Elute the His-tagged protein (up
to 100 mM).
• Avoids hydrophobic interactions
between proteins.
• Up to 50% can be used.
• Coordinates with cations, causing
a decrease in capacity.
• Not recommended, but up to
1 mM in samples has been used
successfully in some cases.
• Avoids hydrophobic interactions
between proteins but may
precipitate proteins, causing
clump clogging and low flow
rates.
• Up to 20% can be used.
• Removes background proteins.
• Up to 2% can be used.
Ni-NTA Agarose Manual EN 201312121403 17

REDUCING AGENTS:
• Reduced
glutathione
• ß-mercaptoethanol
• DTT, DTE
• SDS
• Can reduce Ni 2+ ions at higher
concentrations.
• Up to 30 mM in samples has been
used successfully in some cases.
• Avoids formation of disulfide
bonds. Can reduce Ni 2+ ions at
higher concentrations.
• Up to 20 mM in samples has been
used successfully in some cases.
• Can reduce Ni 2+ ions at higher
concentrations.
• Up to 10 mM in samples has been
used successfully in some cases.
• Avoids hydrophobic interactions
between proteins.
• Coordinates with cations, causing
a decrease in capacity.
• Not recommended, but up to
0.3% in samples has been used
successfully in some cases.
7.2 Tips
• If the lysate is very viscous, 10 µg / ml DNase I (alternatively a mixture of RNase I (5 µg/ml)
und RNase A (10 µg/ml)) may be added.
• Alternatively, pass the solution through a syringe with a wide gauge needle until viscosity is
reduced.
• Under native conditions protease inhibitors such as Pepstatin A, Bestatin, 4-(2-amino-ethyl)
benzene sulphonyl fluoride (AEBSF), E-64 or phosphoramidon may be used.
• Under extended treatments with reducing agents, or in processes where high concentrations of
these reagents are used, reduction of the metal ion may result. This will affect the binding
capacity of the resin, so these agents should be avoided. The reagents described in the
Compatibility Table (see Chapter 7.1) are compatible with Biontex Ni-NTA Agarose Beads under
the conditions and concentrations indicated in this table. The stability of the resin has been
tested in each of the reagents separately.
7.3 Important Information
Biontex Ni-NTA Agarose is developed and sold for research purposes and in-vitro use only. It is not
intended for human therapeutic or diagnostic purposes. Appropriate care should be exercised when
handling many of the reagents described in this publication. Sepharose, ÄKTA, FPLC are registered
trademarks of Amersham Pharmacia Biotech AB.
7.4 Warranty
Biontex guarantees the performance of Biontex Ni-NTA Agarose, when it is used in accordance with
the information given in this publication, for a period of 5 years (after date of certificate of
analysis). If you are not completely satisfied with the performance of the product please contact
Biontex or one of its authorized distributors.
18 Ni-NTA Agarose Manual EN 201312121403

8. Related Products
Product
METAFECTENE ®
EASY+
METAFECTENE ®
SI+
DOTAP
Cat.
No.
T090
T100
T010
Product
Transfection
METAFECTENE ®
PRO
METAFECTENE ®
FluoR
METAFECTENE ®
3D-SC
Cat.
No.
Product
Cat.
No.
T040 METAFECTENE ® T021
T050 INSECTOGENE T030
T110
Protein Purification
Ni-NTA Agarose R040 Ni-IDA Agarose R010
METAFECTENE ®
3D-HG
Highflow Ni-IDA
Agarose
T120
R011
GST Agarose R030 Co-IDA Agarose R020
Highflow Co-IDA
Agarose
R021
Empty S-Columns P-50S Empty L-Columns P-50L Empty XL-Columns P-50XL
Empty Spin-
Columns
SP-50 Empty Cartridges C010
PROTEOfectene ®
E010
Proteofection
PROTEOfectene ®
AB
Cell Culture
E020
MycoSPY ® M030 MycoRAZOR ® M040
µ-Transfection Kit
VI
K010
Microfection Kits
µ-Proteofection Kit
VI
K030
µ-Transfection Kit
VI FluoR
K020
µ-Proteofection Kit
VI AB
K040
Buffer and Positive Controls
1x PBS S011 EASY + buffer (10x) EA010
SI + buffer (10x) SO010 R-Phycoerythrin S020
Visit the Biontex website (http://www.biontex.com) and select your home country to get specific contact and ordering information.
Ni-NTA Agarose Manual EN 201312121403 19

Biontex Laboratories GmbH
Am Klopferspitz 19
im IZB
82152 Planegg/Martinsried
Germany
Phone: +49 (0)89 3247995-0
Fax: +49 (0)89 3247995-2
Fax 2: +49 (0)89 32385994
E-Mail: contact@biontex.com
Internet: www.biontex.com
20 Ni-NTA Agarose Manual EN 201312121403