HIS-Select Technology

True One-Step Histidine-tagged

Protein Purification

 Affinity Resins

 96-Well Plates

 Spin Columns

 Bulk Resins
Purify!
True one-step histidine-tagged protein
purification with Sigma’s HIS-Select Technology ®

■ S
 uperior selectivity for higher purity Sigma’s HIS-Select products purify histidine-tagged proteins quickly
of target proteins and with high selectivity. This is due to Sigma’s patented HIS-Select
■ N
 on-charged, hydrophilic linkage
metal chelate linker, which is hydrophilic and non-charged. Because
reduces non-specific binding
■ O
 ne-step purification
the HIS-Select linkage chemistry is non-charged, nonspecific binding
of unwanted proteins is greatly diminished, allowing for higher purity
of your histidine-tagged protein. Most other immobilized metal
affinity chromatography (IMAC) systems for histidine-tagged protein
purification utilize a charged chelate linkage.

One Step Purification

1 2 3 4 5 6 7 8 9 10 11 In addition, HIS-Select products use a novel tetradentate chelator,
which nearly eliminates metal leaching from the affinity gel, allowing
for higher purity and binding capacity for your target protein. These
technologies combined result in superior selectivity and binding
capacity for your histidine-tagged protein, eliminating the need for
time-consuming secondary purification of your protein and allowing for
one-step-purification.

1
2
TM
Low Range SigmaMarker (M3913)
Whole Cell Extract in Sample Buffer
7
8
Elute 1
Elute 2
For more information, visit sigma.com/hisselect
3 Whole Cell Extract with CelLytic™ B 9 Elute 3
4 Flow Through 10 Elute 4
5 Wash 1 11 Low Range SigmaMarker
6 Wash 2 (M3913)

The figure above depicts a typical purification using

HIS-Select Nickel Affinity Gel. Lanes 7–10 demonstrate
highly purified histidine-tagged protein, eluted with
buffer plus 250 mM imidazole.

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HIS-Select® Products
HIS-Select Nickel Affinity Gel (P6611)
This product is the tool of choice for small to medium scale
purification of histidine-tagged recombinant proteins. The unique,
non-charged, hydrophilic linkage of the nickel chelate group to the
agarose ensures high selectivity for histidine-tagged proteins and
reduces non-specific binding of other proteins and is compatible with
both denaturing or non-denaturing conditions.
HIS-Select HF Nickel Affinity Gel (H0537)
HIS-Select HF (High Flow) Nickel Affinity Gel brings the superior
selectivity of HIS-Select Immobilized Metal Affinity Chromatography
(IMAC) technology to a highly crosslinked agarose designed for higher
flow rates and mechanical stability under pressure. This product is
designed for production scale purification and FPLC™ applications.
As with other HIS-Select products, the non-charged, hydrophilic
linkage of the NTA chelate group to the agarose ensures true one-step
purification.
HIS-Select Cobalt Affinity Gel (H8162)
For those who prefer to use cobalt as the affinity metal ion for
histidine-tagged protein purification, this product allows for high
purity, low non-specific binding, and high binding capacity for your
protein. This product also works well for purification of histidine-
tagged proteins in native, denaturing, or mild reducing conditions.
HIS-Select Spin Columns (H7787)
Prepacked and ready-to-use HIS-Select Spin Columns allow for fast
and consistent small-scale purification of histidine-tagged protein
from crude cell extracts. The Spin Columns contain a matrix of silica
particles and use the patented HIS-Select technology to attain high
protein purity and very low non-specific binding. Spin Columns allow
for fast, one-step purification of at least 150 µg of histidine-tagged
protein in less than 15 minutes.
EZview™ Red HIS-Select HC Nickel Affinity Gel (E3528)
When performing small scale affinity capture, such as
immunoprecipitation, the affinity matrix is difficult to see in the
microcentrifuge tubes. Accidental aspiration of the resin leads to
quantitative variability in results. The EZview Red Affinity Gel greatly
reduces the risk of pellet loss. EZview resins perform as well as
conventional non-colored affinity gels, but allow the user to easily
differentiate pellet from supernatant. This correlates to more accurate
data because less target protein is lost.
HIS-Select High Sensitivity (HS) Nickel Coated Plates
(S5688)
HIS-Select High Sensitivity (HS) Nickel Coated Plates are designed
for highly accurate low-level detection of histidine-tagged proteins.
The captured proteins can be detected using standard enzyme-
linked assay (ELISA) techniques and are suitable for high throughput
applications. These plates are manufactured by utilizing a unique
non-charged hydrophilic linkage for the nickel-chelate, are pre-treated
to reduce non-specific binding, and are sensitive enough to capture as
little as 1 ng/well of histidine-tagged protein. The plates are supplied
as 96-well strip plates.
HIS-Select High Capacity (HC) Nickel Coated Plates (S5563)
HIS-Select High Capacity (HC) Nickel Coated Plates are coated with a
proprietary, high-density nickel chelate matrix. This patented coating
allows for greater per-well binding capacity (≥4 µg/well) than any
other commercial histidine-binding plate and still demonstrates low
non-specific binding. These plates allow for the in-well quantification
of bound protein and are suitable for applications including:
Expression Screening, PAGE, MALDI-MS, and High Throughput
Purification.
HIS-Select iLAP® 5 mL column (H9913)
HIS-Select iLAP (Integrated Lysis and Affinity Purification) columns
are single-use, disposable columns designed for one-step purification
of histidine-tagged proteins directly from a 5 mL bacterial culture.
The single-step method uses our innovative, patent-pending iLAP
technology, which allows quick and simple colony screening for
histidine-tagged recombinant clones. Each column is capable of
purifying at least one milligram of histidine-tagged protein in less than
an hour, resulting in a nearly pure fusion protein preparation following
column elution.
HIS-Select Filter Plate (H0413)
The filter plate is an immobilized metal-ion affinity chromatography
(IMAC) product that incorporates the speed, consistency, and
convenience of spin columns into a multi-sample format. This pre-
packed 96-sample plate has wells that contain 20 µm, spherical silica
particles (100 nm pore size), which are derivitized with a nickel chelate
linkage. The particles allow for small scale histidine-tagged protein
purification from crude cell extracts in less than 15 minutes. The filter
plates are also compatible with centrifugation, vacuum-manifolds,
and robotics systems.
HIS-Select Wash Buffer (H5288)
The buffer is a prepared solution of 10 mM imidazole (in 0.3 M
sodium chloride, and 50 mM sodium phosphate, pH 8.0) that is
optimized to reduce non-specific binding of proteins during wash
steps when purifying histidine-containing proteins using
HIS-Select purification products or other immobilized metal affinity
chromatography (IMAC) products.
HIS-Select Elution Buffer (H5413)
The buffer is a prepared solution of 250 mM imidazole (in 0.3 M
sodium chloride, and 50 mM sodium phosphate, pH 8.0) that is
optimized to elute histidine-containing target proteins from
HIS-Select purification products or other immobilized metal affinity
chromatography (IMAC) products.
HIS-Select® Quick Reference Guide

High Flow (HF) HIS-Select Spin EZview™ Red

Nickel Affinity Gel Nickel Affinity Gel Cobalt Affinity Gel Columns Affinity Gel
Cat. No. P6611 H0537 H8162 H7787 E3528
Package Sizes 5 mL, 25 mL, 100 mL, 10 mL, 25 mL, 100 mL, 5 mL, 25 mL, 100 mL 10 ea., 50 ea. 1 mL, 5 × 1 mL
500 mL 500 mL
Application n  ravity Flow Column
G n F PLC™ n  ravity Flow Column
G n Miniprep n Miniprep Pull-Downs
n Small-Medium Scale n Production Scale n Small-Medium Scale n Process by
Centrifugation
or Vacuum

Scale per Sample 100 µg–10 g 100 µg–100 g 100 µg–10 g 150 µg 10–500 µg per
600 µl/load pull-down
Binding Capacity 15 mg/mL 15 mg/mL 15 mg/mL 150 µg/column 15 mg/mL
Matrix 6% Beaded Agarose 6% Beaded Agarose, 6% Beaded Agarose Silica Particles 6% Beaded Agarose
Highly Cross-linked (Porous, Spherical)
Bead Size 45–165 µm 45–165 µm 45–165 µm ~20 µm 45–165 µm
E xclusion Limit 4 × 10 Da6
4 × 10 Da6
4 × 10 Da6
0.1 µm 4 × 106 Da
(MW) or Pore Size (~10 × 106 Da)
 ax. Linear Flow
M 150 cm/hr 3000 cm/hr 150 cm/hr N/A N/A
Rate (Max. pressure) (5 psi) (200 psi) (5 psi)
 ecommended
R 1 mL/min 5 mL/min 1 mL/min N/A N/A
Flow Rate for
1 × 2 cm Column
 ecommended
R N/A N/A N/A Equivalent to ~80 × g 1 hour at 4 °C
Binding Time/Speed Centrifugation
 ptimal pH
O 3-10 3-10 3-10 3-10 3-10
Stability
Physical Form 50% suspension in 50% suspension in 50% suspension in Prepacked dry matrix in 50% suspension in
30% ethanol 30% ethanol 30% ethanol spin column 30% ethanol

Antimicrobial Agent 30% ethanol 30% ethanol 30% ethanol N/A 30% ethanol
Storage 2-8 °C 2-8 °C 2-8 °C 2-8 °C 2-8 °C
 ecommended
R 0-10 mM 0-10 mM 0-10 mM 0-5 mM 0-10 mM
Imidazole Conc.
for Load/Wash
 ecommended
R 250 mM 250 mM 250 mM 250 mM 250 mM
Imidazole Conc.
for Elution
High Sensitivity
(HS) High Capacity (HC)
Nickel Coated Strip Nickel Coated HIS-Select iLAP® Nickel Filter Plates HIS-Select HIS-Select
Plates Plates 96-well Column 96-well Wash Buffer Elution Buffer
S5688 S5563 H9913 H0413 H5288 H5413
1 ea., 5 ea. 1 ea., 5 × 1 ea. 1 ea., 25 ea. 1 ea., 5 ea. 500 mL, 1 L 250 mL, 500 mL

n  igh Throughput
H n  igh Throughput,
H n  irect Cell Lysis
D n S mall Scale Optimized to reduce Optimized to elute
Screen (ELISA) High Capacity and Purification of n High Throughput non-specific binding of histidine-containing
n Compatible with Screening Histidine-tagged Screening proteins during wash target proteins from
Robotics n Multi-Sample Protein in a Prepacked n Process by steps when purifying HIS-Select purifica-
Miniprep Column Centrifugation or histidine containing tion products or
n In-well Protein Assays n Small Scale Vacuum proteins with HIS-Select other immobilized metal
n Compatible with n Gravity n Compatible with purification products or affinity chromatography
Robotics Robotics other immobilized metal (IMAC) products
affinity chromatography
(IMAC) products
1 ng–1 µg/well 1 –4 µg/well 100 µg–2 mg 1 mg/well N/A N/A
96-well: 200 µl/well 96-well: 200 µl/well 1 mL/load
Sensitivity: ≤1 ng/well 96-well: ≥4 µg/well 2 mg/column 1 mg/well N/A N/A
Clear Polystyrene Plate; Clear Polystyrene Plate; 6% Beaded Agarose Silica Particles N/A N/A
Proprietary Coating Proprietary Coating Tableted Lysis Reagents (Porous, Spherical)
N/A N/A 45–165 µm ~20 µm N/A N/A
N/A ≥300 kDa 4 × 10 Da6
0.1 µm N/A N/A
(~10 × 106 Da)
N/A N/A N/A N/A N/A N/A

N/A N/A Gravity Flow N/A N/A N/A

1 hour at 25 °C 96-well; 4 hours at N/A Equivalent to ~270 × g N/A N/A

25 °C Centrifugation
3-10 3-10 6.0-7.5 3-10 N/A N/A

Clear 96-well flat- Clear 96-well flat- Tablets in 5.5 mL Prepacked dry matrix in Liquid Liquid
bottom, bottom, polystyrene polypropylene column 96-well filter plate
polystyrene strip plate plates
N/A chlorhexidine N/A N/A N/A N/A
2-8 °C 2-8 °C 2-8 °C 2-8 °C Room Temperature Room Temperature
0-5 mM 0-1 mM 0-10 mM 0-5 mM N/A N/A

N/A 200 mM 250 mM 250 mM N/A N/A

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