Clontech Laboratories His60 Ni Superflow Cartridges User Manual - User Manual

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Clontech Laboratories, Inc.

A Takara Bio Company

1290 Terra Bella Avenue, Mountain View, CA 94043, USA

U.S. Technical Support:

tech@clontech.com

United States/Canada

800.662.2566

Asia Pacific

+1.650.919.7300

Europe

+33.(0)1.3904.6880

Japan

+81.(0)77.543.6116

Clontech Laboratories, Inc.

His60 Ni Superflow
Cartridges User
Manual

PT5143-1

Cat. No(s). 635674, 635675, 635678, 635679, 635680

(031716)

His60 Ni Superflow Cartridges User Manual

PT5143-1

www.clontech.com

031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 2 of 15

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

Contact Us

Customer Service/Ordering

Technical Support

tel: 800.662.2566 (toll-free)

fax: 800.424.1350 (toll-free)

web:

www.clontech.com

web:

www.clontech.com

e-mail:

orders@clontech.com

e-mail:

tech@clontech.com

His60 Ni Superflow Cartridges User Manual

PT5143-1

www.clontech.com

031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 3 of 15

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 ml Cartridge

5 ml Cartridge

Support

Superflow 6 (cross-linked agarose)

Bead diameter

–160 μm

Column dimensions (mm i.d.)

0.7 cm x 2.5 cm

1.6 cm x 2.5 cm

Maximum pressure

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 rate

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

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

pH stability short term (≤2 hr)

–

pH stability long term (>2 hr)

–

Binding capacity

60 mg of AcGFPuv

300 mg of AcGFPuv

System compatibility

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

peristaltic pump or syringe

Cartridge body material

Polypropylene

Connectors

1/16” (inlet); 1/16” (outlet)

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.

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

Binding capacity may vary from protein to protein.

Adaptors may be necessary.

His60 Ni Superflow Cartridges User Manual

PT5143-1

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031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 4 of 15

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

ME are required for purification, we strongly recommend using TALON

resin, which provides high

yields of the target protein in up to 30 mM

ME.

His60 Ni Superflow Cartridges User Manual

PT5143-1

www.clontech.com

031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 5 of 15

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

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

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 protein-

dependent.

6 M

Urea

With high concentrations, protein unfolding generally takes

place. Protein refolding on-column (or after elution) is protein-

dependent

Detergents

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.

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.

Tween 20

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

MgCl

4 M

CaCl

5 mM

Ethanol

May precipitate proteins, causing low yields & column clogging.

20%

Glycerol

20%

Detergents cannot be easily removed by buffer exchange.

His60 Ni Superflow Cartridges User Manual

PT5143-1

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031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 6 of 15

II.

List of Components

Store all components at 4

His60 Ni Superflow Cartridges (5 x 1 ml)

(Cat. No. 635675)



5 His60 Ni Superflow Cartridges (1 ml each)



5 Top Caps



5 Snap-Off End Caps

His60 Ni Buffer Set

(Cat. No. 635665)



2 x 250 ml His60 Ni Equilibration Buffer



1 x 200 ml His60 Ni Elution Buffer



1 x 100 ml His60 Ni xTractor

™

Buffer

NOTE:

His60 Ni xTractor Buffer is equivalent to the xTractor Buffer supplied in Cat. Nos. 635623, 635625,

635656 & 635671 (see Section IV).

His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml)

(Cat. No. 635674)



1 His60 Ni Superflow Cartridges (5 x 1 ml) (Cat. No. 635675)



1 His60 Ni Buffer Set (Cat. No. 635665)

His60 Ni Superflow Cartridge (1 x 5 ml)

(Cat. No. 635680)



1 His60 Ni Superflow Cartridge (5 ml each)



1 Top Cap



1 Snap-Off End Cap

His60 Ni Superflow Cartridges (5 x 5 ml)

(Cat. No. 635679)



5 His60 Ni Superflow Cartridges (5 ml each)



5 Top Caps



5 Snap-Off End Caps

His60 Ni Superflow Cartridge Purification Kit (5 x 5 ml)

(Cat. No. 635678)



1 His60 Ni Superflow Cartridges (5 x 5 ml) (Cat. No. 635679)



1 His60 Ni Buffer Set (Cat. No. 635665)

His60 Ni Superflow Cartridges User Manual

PT5143-1

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031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 7 of 15

III.

Additional Materials Required

A. Equipment

For His60 Ni Superflow Cartridges, you will need the following equipment:



A suitable liquid chromatography system (LC procedure only) such as ÄKTA or other systems



A syringe or manual pump

NOTE:

For best results, process all buffers through a 0.45 μm filte

r and degas before use in LC applications.

B. Buffers

– Native Conditions

For your convenience, we provide a separate kit containing a set of His60 Ni Extraction, Equilibration, and

Elution Buffers, the

His60 Ni

Buffer Set

(Cat. No.635665), which is sufficient for approximately 20 purifications

on 1 ml

His60 Ni Superflow Cartridges (5 x 1 ml)

(Cat. No.635675), or 5 purifications on 5 ml

His60 Ni

Superflow Cartridges (5 x 5 ml)

(Cat. No. 635679).

The His60 Ni

Buffer Set is also available together with the 1 ml cartridges in the

His60 Ni Superflow Cartridge

Purification Kit (5 x 1 ml)

(Cat. No. 635674), and together with the 5 ml cartridges in the

His60 Ni Superflow

Cartridge Purification Kit (5 x 5 ml)

(Cat. No. 635678)

—

see Section II.

The following information is provided if you wish to prepare your own buffers for use with other applications.

Please

note that for FPLC and other automated applications, you need to filter the buffers through a 0.45 μm filter

and degas them before use.



Equilibration Buffer

: 50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole; pH 7.4



Wash Buffer:

50 mM sodium phosphate, 300 mM sodium chloride, 40 mM imidazole; pH 7.4

––

Wash Buffer is easily made on a binary pump LC system by mixing 7.1 parts of His60 Ni Elution

Buffer and 92.9 parts of His60 Ni Equilibration Buffer. This buffer ratio can be achieved by running
the LC system at 7.1% Pump B.

––

Alternatively, prepare manually by mixing 710 μl of His60 Ni Elution Buffer with 9.29 ml of His60 Ni

Equilibration Buffer.



Elution Buffer:

50 mM sodium phosphate, 300 mM sodium chloride, 300 mM imidazole; pH 7.4



Regeneration Buffer:

20 mM MES (2-(N-morpholine)-ethanesulfonic acid), 0.3 M sodium chloride; pH

5.0



Imidazole:

Use a highly pure, low-absorbance imidazole ideal for LC applications

(Fisher, Product No. BP 305-50).

His60 Ni Superflow Cartridges User Manual

PT5143-1

www.clontech.com

031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 8 of 15

C. Buffers

– Denaturing Conditions – Guanidine-HCl or Urea

Denaturants, such as 5 M guanidine-HCl or 8 M urea, enhance protein solubility. Because overexpressed proteins

in prokaryotic systems are sometimes insoluble, you may need to purify proteins under denaturing conditions.

When using high concentrations of guanidine-HCl or urea, protein unfolding takes place. On-column refolding or

post-elution refolding is protein-dependent. When purifying proteins under denaturing conditions, we recommend

preparing buffers as indicated below.

Buffers with 6M Guanidine-HCl



Equilibration Buffer

: 50 mM sodium phosphate, 6 M guanidine-HCl, 300 mM NaCl, 20 mM imidazole;

pH 7.4



Wash Buffer:

50 mM sodium phosphate, 6 M guanidine-HCl, 300 mM NaCl, 40 mM imidazole; pH 7.4



Elution Buffer:

50 mM sodium phosphate, 6 M guanidine-HCl, 300 mM NaCl, 300 mM imidazole; pH

7.4

Buffers with 8 M Urea



Equilibration Buffer

: 50 mM sodium phosphate, 8 M urea, 300 mM NaCl, 20 mM imidazole; pH 7.4



Wash Buffer:

50 mM sodium phosphate, 8 M urea, 300 mM NaCl, 40 mM imidazole; pH 7.4



Elution Buffer:

50 mM sodium phosphate, 8 M urea, 300 mM NaCl, 300 mM imidazole; pH 7.4

NOTE:

Samples containing guanidine-HCl cannot be analyzed by SDS-PAGE. A buffer exchange to a buffer

containing urea must be performed before SDS-PAGE analysis. Samples containing urea can be analyzed directly

by SDS-PAGE.

D. Enzymes



Benzonase

(Sigma, Cat. No. E8263-5KU)



Recombinant DNase I

(TaKaRa, Cat. No. 2270A)

E. Accessories for Automated Purification



A suitable liquid chromatography system (LC procedure only) with 1/16 inch tubing

—

or a binary pump

system (a quicker and more convenient alternative)



Additional connectors and fittings required to attach the cartridges to a Bio-Rad BioLogic or a classic
FPLC System (Section III.F)

F. Accessories for Syringe-Based Purification



Luer Lock Syringe Fittings

(GE Healthcare, Cat. No. 18-1112-51) for syringe-based purification only



M6 FPLC Fittings

(GE Healthcare, Cat. Nos. 18-1112-58 & 18-1112-57) for syringe-based or automated

purification

G. Optional



PD-10 desalting columns

(GE Healthcare, Cat. No. 17-0851-01) to remove excess imidazole from the

final sample when required for downstream applications

His60 Ni Superflow Cartridges User Manual

PT5143-1

www.clontech.com

031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 9 of 15

IV.

Related Products: Extraction Buffers, Protease Inhibitors, and His-Tag

Antibodies

xTractor Buffer Kit

(Cat. No. 635623)

Applications:

extraction of insoluble protein from inclusion bodies, efficient extraction of high molecular weight

proteins, and complete disruption of bacterial cell walls and membranes



200 ml xTractor Buffer



400 μl DNase I



2.5 ml Lysozyme

xTractor Buffer

(Cat. Nos. 635656, 635671 & 635625)

Applications:

bacterial lysis, extraction of proteins from yeast cells without the use of glass beads, mammalian

cell pellet extraction, and purification of affinity-tagged proteins



100 ml (Cat. No. 635656)



250 ml (Cat. No. 635671)



500 ml (Cat. No. 635625)

ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail

(Cat. No. 635673)

Complete, easy-to-use protease inhibitor cocktail that is EDTA-free (can be used on IMAC resins without

interfering with protein binding).



10 x 100 μl

(Cat. No. 635673)

Antibodies for detection of tagged proteins



6xHis mAb-HRP conjugate (albumin-free),

100 μl

(Cat. No. 631210)



6xHis Monoclonal Antibody (albumin-free),

200 μg

(Cat. No. 631212)



6xHN Polyclonal Antibody,

200 μl

, (Cat. No. 631213)

His60 Ni Superflow Cartridges User Manual

PT5143-1

www.clontech.com

031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 10 of 15

Sample Preparation & Purification

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING.

Use this procedure to (a) prepare your his-tagged protein sample for (b) automated or (c) manual purification using His60
Ni Superflow Cartridges.

PROTOCOL: Extracting Proteins from Cells

This procedure has been optimized for extraction of proteins from fresh or frozen cell pellets using xTractor
Buffer. The extraction volumes can be adjusted, as long as

20 ml of xTractor Buffer

are used per

1 g of cell

pellet

Resuspend the cell pellet

Add

20 ml of xTractor Buffer

1 g of cell pellet

. Mix gently. Pipet the mixture up and down to

fully resuspend the pellet.

Optional step

– lysozyme & DNase I/protease inhibitor

Add

40 μl of 1 unit/μl DNase I solution

and

200 μl of 100X lysozyme solution

. Add

EDTA-

free protease inhibitor

. Mix gently, pipetting up and down several times.

NOTES:



DNase I reduces the viscosity of the lysate, allowing for more efficient removal of cellular
debris. DNase I can be used without lysozyme. However, if you are treating cells with
lysozyme, you must also treat these cells with DNase I.



Lysozyme helps to fully disrupt bacterial walls and is highly beneficial when extracting high
molecular weight proteins (>40 kDa).



The lysozyme solution may form a precipitate. Resuspend the contents of the bottle and apply

200 μl of suspension directly to

the mix or (optionally) centrifuge 200 μl of

lysozyme

solution for 5 min at 14,000 rpm & use the supernatant to perform the lysis.



We recommend that you use our ProteoGuard EDTA-Free Protease Inhibitor Cocktail
(Cat. Nos. 635672 & 635673).

Incubation

Incubate with gentle shaking for 10 min at room temperature. (If desired, you may incubate the
solution at 4°C.)

NOTE:

At the end of the incubation period, there should be no visible particles. If cell pellet

fragments are present, resuspend them by pipetting the solution up and down and incubating for
an additional 1

–

2 min.

His60 Ni Superflow Cartridges User Manual

PT5143-1

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031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 11 of 15

Lysate clarification

Centrifuge the crude lysate at

10,000–12,000 x g

for

20 min

4°C

. Carefully transfer the

supernatant to a clean tube without disturbing the pellet.

This is your starting sample.

NOTE:

If the supernatant is not clear, centrifuge a second time or filter through a

0.45 µm membrane (e.g., cellulose acetate) to avoid clogging the IMAC column with insoluble
material.

PROTOCOL: Automated Purification on a Liquid Chromatography System

Equilibration

Equilibrate the cartridge and all buffers to

room temperature

4°C

LC System Set up

Prepare the LC system by filling the tubing with buffer. On a binary pump LC system, fill
Pump A with

Equilibration Buffer

and Pump B with

Elution Buffer

Remove the top plug from the cartridge and start pumping

Equilibration Buffer

at a flow

rate of

1 ml/min

until a few drops fill in the top inlet.

Pause the pump, connect the cartridge to the pump outlet, and carefully snap off the bottom
cap of the cartridge (do not twist).

Start the pump. To avoid introducing air into the system, allow a few drops to emerge from
the cartridge before connecting to the LC UV monitor inlet port.

Equilibrate Cartridge

Equilibrate the cartridge with

5–10 column volumes

of the

Equilibration Buffer

at a flow rate

1 ml/min

for

1 ml cartridge

5 ml/min

for

5 ml cartridge

NOTE:

For maximum extraction and binding, prepare the sample using our xTractor Buffer

(Section V.A). If you used incompatible reagents (Section I.B.) during the extraction, desalt the
sample on a PD-10 column (Section III.G) before proceeding to Step 4.

Sample Loading

Load the clarified sample onto the cartridge at a flow rate of

0.5–1 ml/min

and collect fractions.

His60 Ni Superflow Cartridges User Manual

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031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 12 of 15

Wash Step

Wash the cartridge using a flow rate of

1 ml/min

for

1 ml cartridges

5 ml/min

for

5 ml

cartridges

with

8 column volumes

Equilibration Buffer

followed by

7 column volumes

Wash Buffer

(i.e., Equilibration Buffer containing 40 mM imidazole). The Wash Buffer is

prepared on the LC system by running Pump B at 7.1%.

Elution Step

Elute using a flow rate of

1 ml/min

for

1 ml cartridges

5 ml/min

for

5 ml cartridges

with

approximately

5–8 column volumes

Elution Buffer

(containing 300 mM imidazole) and

collect

1 ml fractions

Monitor protein elution by measuring the absorbance of the fractions at 280 nm or performing a
Bradford assay (Bradford, 1976). The collected fractions can be analyzed by SDS-PAGE.

NOTE:

If necessary for downstream applications, remove excess imidazole by gel filtration on a

PD-10 column (Section III.G).

Cartridge Regeneration

Wash cartridge with

20 column volumes

Equilibration Buffer

or wash with

10 column

volumes

20 mM MES, 0.3 M NaCl pH 5.0 buffer

NOTE:

Regeneration allows the cartridge to be reused to purify the same protein, without

significant loss of binding capacity, up to 5 times depending on the purification conditions and
the target protein.

Extended Storage (over 1 week)

Wash the cartridge with

five column volumes

water

after each use. Store in

20% ethanol

4°C Attach supplied bottom cap, followed by the top plug.

C. PROTOCOL: Manual Purification Using a Syringe

Equilibration

Equilibrate the cartridge and all buffers to

room temperature

or at

4°C

Luer Lock Syringe Setup

Fill syringe with

5–10 column volumes

Equilibration Buffer

His60 Ni Superflow Cartridges User Manual

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Clontech Laboratories, Inc. A Takara Bio Company

Page 13 of 15

Cartridge Setup

Attach the syringe to a Luer Lock Adapter (not supplied; see Section III.F).

Remove the top plug from the cartridge, add a few drops of buffer from the syringe to the top
inlet of the cartridge, and attach the syringe to the top of the cartridge via the Luer Lock
adapter.

Carefully snap off the bottom cap of the cartridge (do not twist).

Equilibrate Cartridge

Equilibrate the cartridge with

5–10 column volumes

of the

Equilibration Buffer

at a flow rate

1 ml/min

for

1 ml cartridge

5 ml/min

for

5 ml cartridge

Remove the syringe from the Luer Lock Adapter.

NOTE:

For maximum extraction and binding, prepare the sample using our xTractor Buffer

(Section V.A). If you used incompatible reagents (Section I.B.) during the extraction, desalt the
sample on a PD-10 column (Section III.G) before proceeding to Step 5.

Sample Loading

Fill the syringe with the clarified sample and reconnect it to the Luer Lock Adapter. Slowly push
the syringe plunger to pass the sample through the cartridge. For maximum binding and better
yields, load the sample at an approximate flow rate

of 0.5–1 ml/min

and collect fractions.

Wash Step

Using a clean syringe, wash the resin with

10 column volumes

Wash Buffer

at a flow rate of

~1 ml/min

for

1 ml cartridges

~5 ml/min

for

5 ml cartridges

NOTE:

If you are using the buffers supplied in the

His60 Ni Buffer Set

(Cat. No. 635665), the

His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml)

(Cat. No. 635674), or the

His60 Ni

Superflow Cartridge Purification Kit (5 x 5 ml)

(Cat. No. 635678), prepare the

Wash Buffer

by mixing

7.1 parts

Elution Buffer

with

92.9 parts

Equilibration Buffer

Elution Step

Using a clean syringe, elute the sample at a flow rate of

~1 ml/min

for

1 ml cartridges

~5 ml/min

for

5 ml cartridges

with approximately

five column volumes

Elution Buffer

collecting 1 ml fractions.

Monitor protein elution by measuring the absorbance of the fractions at 280 nm or performing a
Bradford assay (Bradford, 1976). The collected fractions can be analyzed by SDS-PAGE.

His60 Ni Superflow Cartridges User Manual

PT5143-1

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031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 14 of 15

NOTE:

If necessary for downstream applications, remove excess imidazole by gel filtration on a

PD-10 column (Section III.G).

Cartridge Regeneration

Wash cartridge with

20 column volumes

Equilibration Buffer

or wash with

10 column

volumes

20 mM MES, 0.3 M NaCl pH 5.0 buffer

NOTE:

Regeneration allows the cartridge to be reused to purify the same protein, without

significant loss of binding capacity, up to 5 times depending on the purification conditions and
the target protein.

Extended Storage (over 1 week)

Wash the cartridge with

five column volumes

water

after each use. Store in

20% ethanol

4°C Attach supplied bottom cap, followed by the top plug.

D. PROTOCOL: Complete Regeneration of His60 Ni Superflow Cartridges

If you plan to purify multiple proteins using the same column, you must use the following resin regeneration protocol

before you purify a new protein.

Strip Ni ions

Wash the cartridge with

ten column volumes

0.2 M EDTA (pH 7.0)

at room temperature.

Wash excess EDTA from the cartridges with an additional

ten column volumes

double

distilled H

O (ddH

Charge Resin

Add

two column volumes

100 mM NiSO

solution

Wash resin to remove excess ions with

seven column volumes

ddH

, followed by

3 column

volumes

300 mM NaCl

and

3 column volumes

ddH

Equilibrate Resin

Add

ten column volumes

Equilibration/Wash Buffer

Cartridge is ready to use

Resin can be regenerated up to 5 times.

His60 Ni Superflow Cartridges User Manual

PT5143-1

www.clontech.com

031716

Clontech Laboratories, Inc. A Takara Bio Company

Page 15 of 15

VI.

Troubleshooting Guide

Table 3. Troubleshooting Guide for His60 Ni Superflow Cartridges

Description of Problem

Possible Explanation

Solution

Low target yield

Poor expression of target protein

Optimize bacterial expression

conditions.

Target protein forms inclusion bodies



Decrease temperature to 25°C or

lower during induction to

minimize inclusion body

formation.



Solubilize inclusion bodies and

perform the purification in the

presence of 8 M urea or 6 M

guanidinium HCl.

Inefficient target extraction

Use our xTractor Buffer.

Inaccessible polyhistidine tag

Purify in presence of 6

–

8 M urea or

6 M guanidinium HCl.

Impurities in eluate

Insufficient washing

Increase wash volume or add

intermediate wash at 60 mM

imidazole. (This can result in partial

loss of target protein.)

Low flow rate

Clogged cartridge

Apply only clarified extract, and

decrease the amount of loaded

sample.

Viscous sample

Treat sample with Benzonase

DNase I, as described in Section V.A.

Can not detect target protein by
UV



Use low UV absorbance

imidazole in the buffers.



Perform a Bradford protein assay

on collected fractions to identify

target protein in eluate.

Notice to Purchaser

Our products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs,

in vitro

diagnostic

purposes, therapeutics, or in humans. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to

provide a service to third parties without prior written approval of Clontech Laboratories, Inc.

Your use of this product is also subject to compliance with any applicable licensing requirements described on the product’s

web page at

http://www.clontech.com

. It is

your responsibility to review, understand and adhere to any restrictions imposed by such statements.
Clontech®, the Clontech logo, ProteoGuard, TALON®, and xTractor are trademarks of Clontech Laboratories, Inc. All other trademarks are the property of their

Inc.
This document has been reviewed and approved by the Clontech Quality Assurance Department.

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