Clontech Laboratories His60 Ni Superflow Cartridges User Manual

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Clontech Laboratories, Inc.

His60 Ni Superflow
Cartridges User
Manual

PT5143-1
Cat. No(s). 635674, 635675, 635678, 635679, 635680
(031716)

Clontech Laboratories, Inc.

A Takara Bio Company
1290 Terra Bella Avenue, Mountain View, CA 94043, USA
U.S. Technical Support: [e-mail]

His60 Ni Superflow Cartridges User Manual

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Table of Contents

I. Introduction ..................................................................................................................................................................... 3

A. His60 Ni Superflow 1 ml & 5 ml Cartridge Specifications ........................................................................................ 3

B. His60 Ni Superflow Resin .......................................................................................................................................... 4

II. List of Components ......................................................................................................................................................... 6

III. Additional Materials Required .................................................................................................................................... 7

A. Equipment ................................................................................................................................................................... 7

B. Buffers – Native Conditions ....................................................................................................................................... 7

C. Buffers – Denaturing Conditions – Guanidine-HCl or Urea....................................................................................... 8

D. Enzymes ...................................................................................................................................................................... 8

E. Accessories for Automated Purification ..................................................................................................................... 8

F. Accessories for Syringe-Based Purification ................................................................................................................ 8

G. Optional ....................................................................................................................................................................... 8

IV. Related Products: Extraction Buffers, Protease Inhibitors, and His-Tag Antibodies ................................................. 9

V. Sample Preparation & Purification ............................................................................................................................... 10

A. PROTOCOL: Extracting Proteins from Cells ........................................................................................................... 10

B. PROTOCOL: Automated Purification on a Liquid Chromatography System .......................................................... 11

C. PROTOCOL: Manual Purification Using a Syringe ................................................................................................. 12

D. PROTOCOL: Complete Regeneration of His60 Ni Superflow Cartridges ............................................................... 14

VI. Troubleshooting Guide ............................................................................................................................................. 15

Table of Tables

Table 1. His60 Ni Superflow Cartridge Specifications ........................................................................................................... 3

Table 2. Reagent Compatibility with His60 Ni Superflow Resin (Based on Literature References) ..................................... 5

Table 3. Troubleshooting Guide for His60 Ni Superflow Cartridges ................................................................................... 15

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His60 Ni Superflow Cartridges User Manual

1 ml Cartridge

5 ml Cartridge

Support

Superflow 6 (cross-linked agarose)

Bead diameter

60–160 μm

Column dimensions (mm i.d.)

0.7 cm x 2.5 cm

1.6 cm x 2.5 cm

Maximum pressure1

5 bar, 0.5 MPa

Typical back pressure

1.0 bar, 0.1 MPa (1 ml/min)

2.0 bar, 0.2 MPa (5 ml/min)

Recommended flow rate

1 ml/min (156 cm/hr)

5 ml/min (149 cm/hr)

Maximum flow rate2

10 ml/min (1,559 cm/hr)

40 ml/min (1,193 cm/hr)

pH stability short term (≤2 hr)

2–14

pH stability long term (>2 hr)

3–14

Binding capacity3

60 mg of AcGFPuv

300 mg of AcGFPuv

System compatibility4

Automated chromatography systems (e.g., ÄKTA, FPLC, etc.),

peristaltic pump or syringe

Cartridge body material

Polypropylene

Connectors

1/16” (inlet); 1/16” (outlet)

I. Introduction

Clontech’s His60 Ni Superflow Cartridges are prefilled with 1 ml or 5 ml of His60 Ni Superflow resin and are
ready to use for purification of his-tagged proteins using a syringe, peristaltic pump, or liquid chromatography
systems such as ÄKTA or other FPLC systems.

A. His60 Ni Superflow 1 ml & 5 ml Cartridge Specifications

Table 1. His60 Ni Superflow Cartridge Specifications

1

The maximum pressure that can be used with the Superflow matrix itself is 10 bar. However, stability of the cartridges is

only guaranteed up to 5 bar.

2

High flow rates may lead to reduced recovery of 6xHis-tagged protein.

3

Binding capacity may vary from protein to protein.

4

Adaptors may be necessary.

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His60 Ni Superflow Cartridges User Manual

B. His60 Ni Superflow Resin

Clontech’s His60 Ni Superflow Resin is a high-capacity Ni-IDA resin that has been optimized for the efficient
one-step purification of expressed his-tagged proteins from bacterial, yeast, mammalian, and baculovirus-infected
cells. The combination of the high density of nickel (II) ion and the high flow rates of the Superflow 6 agarose
beads allow the efficient capture of target his-tagged proteins. Up to 60 mg of his-tagged protein can be adsorbed
onto 1 ml of His60 Ni resin (data based on purification of AcGFP1).

The His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml) provides 5 prepacked His60 Ni Superflow
Cartridges (each containing 1 ml of resin), as well as all the buffers needed for protein extraction and purification
(also available separately as the His60 Ni Buffer Set)—see Section II.

The His60 Ni Cartridge Purification Kit (5 x 5 ml) provides 5 prepacked His60 Ni Superflow Cartridges
(each containing 5 ml of resin), as well as all the buffers needed for protein extraction and purification (also
available separately as the His60 Ni Buffer Set)—see Section II.

Limitations of His60 Ni Superflow Resin

Please note the following recommendations when using His60 Ni Superflow Cartridges:

 Do not use chelator-containing protease inhibitors or other additives, EDTA, or strong reducing agents

(see Table 2 and the note below regarding the use of reducing agents).

 For automated liquid-chromatography (LC) applications, use highly pure, low-absorbance imidazole

(Fisher, Product No. BP 305-50). Always filter buffers through a 0.45 μm filter and degas before use.

 His60 Ni allows protein purification under either native or denaturing conditions. The resin is compatible

with multiple denaturants and detergents (Table 2).

NOTE: Using βME as a reducing agent with His60 Ni Superflow Resin sharply reduces protein yield. If high
levels of βME are required for purification, we strongly recommend using TALON resin, which provides high
yields of the target protein in up to 30 mM βME.

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His60 Ni Superflow Cartridges User Manual

Reagent

Notes

Acceptable Concentrations

Amino Acids

Arginine, Glycine,

Glutamine

Not recommended

Histidine

Binds to His60 Ni and competes with histidine residues in the
his-tag.

Can be used at low concentrations (20

mM) to inhibit nonspecific binding;

and, at a higher concentration (up to

100 mM), to elute his-tagged proteins

Buffers

HEPES, MOPS

Amine groups that are present in these buffers can interact with
Ni2+ ions, diminishing the resin’s binding capacity.

Up to 100 mM (with caution)

Sodium acetate

Up to 100 mM (with caution)

Sodium phosphate

Up to 50 mM can be used

Tris

Coordinates weakly with metal ions, causing a decrease in
binding capacity.

Up to 50 mM (with caution). Loss in

binding capacity can be seen.

Chelating Agents

EDTA, EGTA

Will strip metal ions from the resin, resulting in protein elution
and a resin color change.

Not recommended

Denaturing Agents

Gu-HCl

With high concentrations, protein unfolding generally takes
place. Protein refolding on-column (or after elution) is protein­dependent.

6 M

Urea

With high concentrations, protein unfolding generally takes
place. Protein refolding on-column (or after elution) is protein­dependent

8M

Detergents1

CHAPS

Ionic detergents such as CHAPS, SDS, and sarkosyl are
compatible at concentrations up to 1%. Even at low
concentrations you should expect interference with binding.

1% (with caution)

NP-40

Has high absorbance at 280 nm.

2%

SDS

Ionic detergents such as CHAPS, SDS, and sarkosyl are
compatible at concentrations up to 1%. Even at low
concentrations you should expect interference with binding.

1% (with caution)

Triton X-100

Has high absorbance at 280 nm.

1%

Tween 20 2%

Reducing Agents

ßME

Use the resin immediately after equilibrating with buffers

containing βME. Otherwise the resin will change color. Do not
store the resin in buffers containing βME. A slight change in

color (yellowing of the resin) will occur.

20 mM (with caution)

DTT

Since DTT is a reducing agent, low concentrations will reduce
the metal ions in His60 Ni Superflow resin. Although enough of
these ions may remain unaffected to allow protein purification,
please use it with caution. Do at least 20 column volumes of
washes, preferably with low concentrations of imidazole (40
mM) to wash out any reduced metal ions.

1 mM (with caution)

DTE Not recommended

Others

MgCl2

4 M

CaCl2

5 mM

Ethanol

May precipitate proteins, causing low yields & column clogging.

20%

Glycerol

20%

1

Detergents cannot be easily removed by buffer exchange.

Table 2. Reagent Compatibility with His60 Ni Superflow Resin (Based on Literature References)

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