Selection guide
Total solutions
for preparation of
histidine-tagged
proteins
2
Cytiva offers a wide range of products for convenient and flexible purification of
histidine-tagged proteins produced in prokaryotic and eukaryotic expression systems.
Five categories of chromatography media (resins) are available — Ni Sepharose™
High Performance (HP), Ni Sepharose 6 Fast Flow (FF), His Mag Sepharose Ni, which
are precharged with nickel ions, TALON
®
Superflow™ (precharged with cobalt ions),
Ni Sepharose excel, His Mag Sepharose excel (precharged with nickel ions very strongly
bound to the chelating ligand), and uncharged IMAC Sepharose HP and IMAC Sepharose
6 FF. These media are available in a range of prepacked formats for convenience.
Cytiva’s products for histidine-tagged protein purification offer the following benefits:
• Flexible choice of uncharged or nickel/cobalt precharged media
• High protein binding capacity with low leakage of nickel or cobalt ions
• Range of products covers purification of tagged proteins from prokaryotic and
eukaryotic expression systems
• Different formats allow screening, capture purification, and scale-up
Use the workflow on page 3 to locate the right product for your needs on the pages
of this guide.
3
Select your product category
How is the protein expressed?
Extracellular
Intracellular
What are your
requirements?
High purity
High capacity
Precharged
TALON Superflow
(cobalt-based)
products
(page 9)
Precharged
Ni Sepharose excel
products
(page 7)
Precharged
Ni Sepharose
products
(page 4)
Uncharged
IMAC Sepharose
products
(page 11)
Which
expression
system are you
using?
Prokaryotic
Eukaryotic
4
Ni Sepharose High Performance (HP), Fast Flow (FF), and His Mag Sepharose Ni media are precharged with Ni
2+
ions for
convenience. These media provide high binding capacity combined with lower leakage of Ni
2+
ions compared to products
from other suppliers
*
, which ensures reliable capture of histidine-tagged proteins with high repeatability between runs.
Ni Sepharose HP has a high protein binding capacity and the small bead size ensures high-performance purification
and minimal sample dilution. Ni Sepharose HP is available in lab packs and prepacked HisTrap™ HP columns, as well as
His SpinTrap™ columns for small-scale purification (minipreps) by centrifugation and His MultiTrap™ HP 96-well plates
for high-throughput screening and parallel purification.
Ni Sepharose 6 FF has high protein binding capacity, which converts to lower costs per prep as a result of the lower
medium requirement. The flow properties of the highly cross-linked agarose matrix make it an excellent choice for
purification scale-up.
Ni Sepharose 6 FF is available in bulk packs for batch purification and scale-up. The medium is also available in prepacked
His MultiTrap FF 96-well filter plates for screening purposes and His GraviTrap™ columns for convenient and reproducible
gravity-flow preparations. To enable reliable scale-up, Ni Sepharose 6 FF is available in HisTrap FF columns that can be
connected in series, as well as larger HisPrep™ FF 16/10 columns when greater capacity is needed. Purification can be
performed directly from unclarified lysates using HisTrap FF crude, His GraviTrap, His SpinTrap, and His MultiTrap columns
and 96-well plates. This allows fast purifications and reduces sample preparation time, which minimizes degradation of
sensitive target proteins by proteases present in the cell lysate. For process development, prepacked HiScreen™ Ni FF
columns with 10 ml bed heights are used. These columns can be connected in series for increased capacity.
His Mag Sepharose Ni consists of Sepharose based magnetic beads designed for small-scale purification and expression
screening of histidine-tagged proteins. These visible and dense beads allow convenient capture and simplified handling of
histidine-tagged proteins using a magnetic rack.
Ni Sepharose excel and His Mag Sepharose excel are IMAC media designed for screening and purification of
histidine-tagged proteins secreted into eukaryotic cell culture supernatants. Find out more about Ni Sepharose excel
and His Mag Sepharose excel on page 7.
* See Data files 18-1174-40, 11-0008-86, 28-4041-05, 28-9797-23, and 28-4041-06 for details.
Precharged Ni Sepharose products
5
Product
Product
code no.
Data le
code no. Pack size
Approx. protein
binding capacity
*
High-
throughput
screening Minipreps
Batch/Gravity
ow
Syringe
compatible Scale-up
ÄKTA™ system
compatibility
Process
development
Magnetic
separation
Ni Sepharose
High Performance
17-5268-01 18-1174-40 25 ml 40 mg/ml • •
17-5268-02 100 ml • •
His MultiTrap HP 28-4009-89 11-0036-63 4 × 96-well lter plates 1000 μg/well • •
His SpinTrap 28-4013-53 28-4046-59 50 × 100 μl 750 μg/column • •
His SpinTrap Kit
28-9321-71 28-4046-59 50 × 100 µl 750 µg/column • •
HisTrap HP 17-5247-01 18-1174-40 5 × 1 ml 40 mg/column •
17-5247-05 100 × 1 ml
§
•
17-5248-01 18-1174-40 1 × 5 ml 200 mg/column •
17-5248-02 5 × 5 ml •
17-5248-05 100 × 5 ml
§
•
Ni Sepharose 6 Fast Flow 17-5318-06 11-0008-86 5 ml 40 mg/ml • • • • •
17-5318-01 25 ml • • • • •
17-5318-02 100 ml • • • • •
17-5318-03 500 ml • • • • •
His Mag Sepharose Ni 28-9673-88 2 × 1 ml 50 mg/ml • •
28-9673-90 5 × 1 ml • •
28-9799-17 10 × 1 ml • •
His MultiTrap FF 28-4009-90 11-0036-63 4 × 96-well lter plates 800 μg/well • •
His GraviTrap 11-0033-99 11-0036-90 10 × 1 ml 40 mg/column •
His GraviTrap Kit
‡
28-4013-51 11- 0036-90 20 × 1 ml 40 mg/column •
HisTrap FF 17-5319-01 11-0008-86 5 × 1 ml 40 mg/column • •
17-5319-02 100 × 1 ml
§
• •
17-5255-01 11-0008-86 5 × 5 ml 200 mg/column • •
17-5255-02 100 × 5 ml
§
• •
HisTrap FF crude 11-0004-58 11-0012-37 5 × 1 ml 40 mg/column • •
11-0004-59 100 × 1 ml
§
• •
17-5286-01 11-0012-37 5 × 5 ml 200 mg/column • •
17-5286-02 100 × 5 ml
§
• •
HisTrap FF crude Kit
†
28-4014-77 11-0012-37 3 × 1 ml 40 mg/column •
HiScreen Ni FF 28-9782-44 28-9305-81 1 × 4.7 ml 40 mg/column • • •
HisPrep FF 16/10 28-9365-51 11-0008-86 1 × 20 ml 800 mg/column • •
Buffer kit
His Buffer Kit 11-0034-00 11- 0036-90 • • •
* Protein-dependent
†
Includes buffer stock solutions for approximately 10 preps using a syringe
‡
Includes buffer stock solutions for approximately 20 preps
Includes buffer stock solutions for approximately 50 preps
§
Available by specic customer order
Technical overview
6
Selecting Ni Sepharose
products for manual/
automated purification
Will you use
an automated
purification
system?
Ni Sepharose High
Performance
Ni Sepharose
6 Fast Flow
His MultiTrap FF
His MultiTrap HP
His GraviTrap His GraviTrap Kit
His Mag
Sepahrose Ni
His SpinTrap Kit
His SpinTrap
Ni Sepharose
6 Fast Flow
HisTrap HP HiScreen Ni FF
Process
development
What are your
requirements?
Yes
Yes
Yes
No
No
No
No Yes
Yes
Yes
No No
HisPrep FF 16/10HisTrap FF HisTrap FF crude
High resolution
Scalability/high
flow rates
Prepacked
columns?
Prepacked
columns?
Gravity flow
column
Buffers included? Magnetic beads
Spin column
96-well plate
Batch
Syringe
Buffers included?
HisTrap FF
crude Kit
HisTrap FF
< 200 mg
Buffers included?
Clarified or
unclarified sample
Amount of histidine
tagged protein
> 200 mgUnclarified
Clarified
7
Ni Sepharose excel products
Purification from eukaryotic cell culture
supernatants
Traditionally, IMAC purification of histidine-tagged proteins secreted
into eukaryotic cell culture supernatants often leads to stripping of the
immobilized metal ions during sample application. This results in low
or no binding of the target protein. Purification is further complicated
by the fact that the target protein concentration is low, which results
in large sample volumes. To overcome these problems, extensive
sample pretreatment is required, such as buffer exchange by dialysis in
combination with concentration procedures. Such pretreatment is often
time-consuming and potentially harmful to sensitive proteins.
Ni Sepharose excel is a novel IMAC medium precharged with nickel ions
that are bound very strongly. Ni Sepharose excel is designed primarily
for capture and purification of histidine-tagged proteins secreted into
eukaryotic cell culture supernatants, such as from insect cells or CHO
cells. The medium is also suitable for purification of histidine-tagged
proteins originating from other samples causing nickel stripping. Ni
Sepharose excel is available in lab packs for scale-up and packing in
chromatography columns such as Tricorn™, XK, or HiScale™ columns as
well as in prepacked HisTrap excel columns, which allow purification of
unclarified lysates when used with chromatography systems.
His Mag Sepharose excel are Sepharose based magnetic beads
designed for small-scale purification and expression screening
of histidine-tagged proteins. These visible and dense beads allow
convenient capture and handling of histidine-tagged proteins using
a magnetic rack.
Product
Product
code no.
Data le
code no.
Pack
size
Approx. protein
binding capacity*
High-
throughput
screening Minipreps
Batch/Gravity
ow
Syringe
compatible Scale-up
ÄKTA system
compatibility
Ni Sepharose excel 17-3712-01 29-0168-49 25 ml 10 mg/ml • • •
17-3712-02 100 ml • • •
17-3712-03 500 ml • • •
HisTrap excel 17-3712-05 29-0168-49 5 × 1 ml 10 mg/column • •
17-3712-06 5 × 5 ml 50 mg/column • •
His Mag Sepharose excel 17-3712-20 29-0168-49 2 × 1 ml 10 mg/ml • •
17-3712-21 5 × 1 ml • •
17-3712-22 10 × 1 ml • •
* Protein-dependent
Technical overview
8
Prokaryotic
Selecting Ni Sepharose excel products
Precharged
TALON Superflow
(cobalt-based)
products
(page 9)
Ni Sepharose excel
His Mag
Sepharose excel
HisTrap excel
Eukaryotic
Prepacked
columns?
Uncharged
IMAC Sepharose
products
(page 11)
Cytoplasmic
expression
Precharged
Ni Sepharose
products
(page 4)
Will you use an automated
chromatography system?
Which
expression
system are you
using?
How is the protein
expressed?
Periplasmic/extracellular
secretion
Yes No
Yes No
9
TALON Superflow is a cobalt-based IMAC medium offering
an alternative selectivity compared to nickel-charged media. It is
the first-choice precharged medium when target protein purity is
more important than target protein yield. TALON Superflow binds
polyhistidine-tagged proteins with enhanced selectivity and exhibits
a reduced affinity for host proteins giving lower background. The
medium is suitable for IMAC purification of histidine-tagged proteins
when Ni
2+
is not the optimal choice of metal ion. It is appropriate for
purifying proteins under native or denaturing conditions and allows
use of imidazole concentrations in sample, wash and elution buffers
that are typically lower than those used with nickel-based media.
TALON Superflow is available in lab packs for batch purification and
scale-up and in versatile prepacked formats including His MultiTrap
TALON 96-well filter plates for screening/parallel purification
,
His SpinTrap TALON for minipreps, and His GraviTrap TALON columns
for reproducible gravity-flow preparations. HisTrap TALON crude
columns enable direct loading of unclarified cell lysates for fast and
convenient purification of histidine-tagged proteins.
Precharged TALON Superflow
(cobalt-based) products
Product
Product
code no.
Data le
code no.
Pack
size
Approx. protein
binding capacity*
High-
throughput
screening Minipreps
Batch/
Gravity ow
Syringe
compatible Scale-up
ÄKTA system
compatibility
TALON Superow 28-9574-99 29-0168-49 10 ml 20 mg/ml • • •
28-9575-02 50 ml 20 mg/ml • • •
His MultiTrap TALON 29-0005-96 28-9664-10 4 × 96-well
plates
up to 1 mg/well • •
His SpinTrap TALON 29-0005-93 28-9664-10 50 × 100 μl up to 1 mg/column • •
His GraviTrap TALON 29-0005-94 28-9664-10 10 × 1 ml up to 15 mg/column •
HiTrap™ TALON crude 28-9537-66 28-9664-10 5 × 1 ml 20 mg/column • •
28-9538-05 100 × 1 ml
†
20 mg/column • •
28-9537-67 5 × 5 ml 100 mg/column • •
28-9538-09 100 × 5 ml
†
100 mg/column • •
*
Protein-dependent
†
Available by specic customer order
Technical overview
10
Will you use an automated
chromatography system?
TALON Superflow
His SpinTrap TALONHiTrap TALON crude His GraviTrap TALON
TALON Superflow
HiTrap TALON crude
His MultiTrap TALON
Precharged Ni Sepharose
products (page 4)
Uncharged
IMAC Sepharose
products (page 11)
NoYes
Syringe
Batch
96-well plate Gravity flow column
What are your
requirements?
Selecting TALON Superflow products
for manual/automated purification
Spin column
No
Yes
Prepacked columns?
High protein
purity
High protein
binding
capacity
11
Uncharged IMAC Sepharose products
Immobilized metal ion affinity chromatography (IMAC) exploits a molecule’s affinity for chelated metal ions. The
amino acid histidine present in many proteins forms complexes with transition metal ions such as Co
2+
, Cu
2+
, Zn
2+
,
Ni
2+
, and Fe
3+
. Media with a suitable immobilized metal ion will therefore selectively retain proteins with exposed
histidine residues. Exposed cysteine and tryptophan residues may also be involved in the binding to an immobilized
metal ion but their contribution to the binding is much lower than the contribution from exposed histidine residues.
IMAC Sepharose HP medium is supplied free of metal ions. The medium can be charged with a metal ion of choice,
which allows screening to select the most suitable metal ion and purification conditions for specific histidine-
tagged and untagged target proteins. IMAC Sepharose HP ensures low sample dilution and excellent separation.
The small bead size of the Sepharose HP matrix allows high-performance purifications. The medium is available in
lab packs and in prepacked HiTrap IMAC HP columns.
IMAC Sepharose 6 FF is an uncharged medium for screening for optimal purification conditions and scaling up
purification of histidine-tagged and untagged proteins. The medium is supplied ready for charging with metal
ions. The excellent flow properties of IMAC Sepharose 6 FF allow easy scale-up and the medium is also available in
convenient, prepacked HiTrap IMAC FF columns. Further scale-up can be achieved with HiPrep™ IMAC FF 16/10,
20 ml columns. For process development, prepacked HiScreen IMAC FF columns with 10 ml bed heights are used.
These columns can be connected in series for increased capacity.
12
Product
Product
code no.
Data le
code no. Pack size
Approx. protein
binding capacity*
High-
throughput
screening Minipreps
Batch/Gravity
ow
Syringe
compatible Scale-up
ÄKTA system
compatibility
Process
development
Uncharged IMAC Sepharose
IMAC Sepharose High
Performance
17-0920-06 28-4041-05 25 ml 40 mg/ml (Ni
2+
)* • • •
17-0920-07 100 ml • • •
HiTrap IMAC HP 17-0920-03 28-4041-05 5 × 1 ml 40 mg/column (Ni
2+
)* • •
17-0920-05 5 × 5 ml 200 mg/column (Ni
2+
)* • •
IMAC Sepharose 6 Fast Flow 17-0921-07 28-4041-06 25 ml 40 mg/ml (Ni
2+
)* • • • • •
17-0921-08 100 ml 25 mg/ml (Cu
2+
)* • • • • •
15 mg/ml (Zn
2+
)*
HiTrap IMAC FF 17-0921-02 28-4041-06 5 × 1 ml 40 mg/column (Ni
2+
)* • • •
17-0921-04 5 × 5 ml 200 mg/column (Ni
2+
)* • • •
125 mg/column (Cu
2+
)*
75 mg/column (Zn
2+
)*
HiPrep IMAC FF 16/10 28-9365-52 28-4041-06 1 × 20 ml 800 mg/column (Ni
2+
)* • •
500 mg/column (Cu
2+
)*
300 mg/column (Zn
2+
)*
HiScreen IMAC FF 28-9505-17 28-9305-81 1 × 4.7 ml 40 mg/column (Ni
2+
)* • • •
15 mg/column (Zn
2+
)**
25 mg/column (Cu
2+
)**
Buffer kit
His Buffer Kit 11-0034-00 11-0036-90 • • •
* (Histidine)6-tagged proteins, protein-dependent
**Untagged protein, protein-dependent
Technical overview
13
Selecting IMAC Sepharose products
for manual/automated purification
Will you use
an automated
purification
system?
No
Yes
IMAC Sepharose
6 Fast Flow
HiPrep IMAC FF 16/10 HiTrap IMAC FFHiTrap IMAC HP
IMAC Sepharose
High Performance
Prepacked columns?
What are your
requirements?
< 200 mg> 200 mg
Yes No
Process development
Scalability/
high flow rates
Prepacked columns?
HiScreen IMAC FF
Yes
No
Gravity flow/batch Syringe
HiTrap IMAC FF
IMAC Sepharose 6
Fast Flow
Amount of histidine
tagged protein
High resolution
cytiva.com/protein-purification
Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or an
affiliate. ÄKTA, GraviTrap, HiPrep, HiScale, HiScreen, HisPrep, HisTrap, HiTrap, MultiTrap,
SpinTrap, Sepharose, and Tricorn are trademarks of Global Life Sciences Solutions USA
LLC or an affiliate doing business as Cytiva.
Superflow is a trademark of Sterogene Bioseparations Inc. TALON is a registered
trademark of Clontech Inc. All third party trademarks are the property of their
respective owners.
IMAC Sepharose products and Ni Sepharose products. Purification and preparation
of fusion proteins and affinity peptides comprising at least two adjacent histidine
residues may require a license under US patent numbers 5,284,933 and 5,310,663,
and equivalent patents and patent applications in other countries (assignee: Hoffman
La Roche, Inc). IMAC Sepharose products, Ni Sepharose products and Fe Sepharose
products.These products are sold under a license from Sigma-Aldrich under patent
number EP 1277616 (Metal chelating compositions) and equivalent patents and patent
applications in other countries.
© 2020 Cytiva
All goods and services are sold subject to the terms and conditions of sale of the
supplying company operating within the Cytiva business. A copy of those terms and
conditions is available on request. Contact your local Cytiva representative for the most
current information.
For local office contact information, visit cytiva.com/contact
CY12758-27Aug20-SG