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