HisTrap HP, 1 ml and 5 ml

HisTrap

HP, 1 ml and 5 ml

Instructions for Use

HisTrap™ HP is a ready to use HiTrap™ column, prepacked with precharged

Ni Sepharose™ High Performance. This prepacked column is ideal for

preparative purification of histidine-tagged recombinant proteins by

immobilized metal ion affinity chromatography (IMAC).

The special design of the column, together with the high-performance

matrix of the Ni Sepharose medium, provides fast, simple, and easy

separations in a convenient format.

Ni Sepharose High Performance has low nickel (Ni

) ion leakage and is

compatible with a wide range of additives used in protein purification.

HisTrap HP columns can be operated with a syringe, peristaltic pump, or

liquid chromatography system such as ÄKTA™.

cytiva.com

71502768 AL

Table of Contents

Product description .............................................................................. 3

2 General considerations ....................................................................... 7

3 Preparation .............................................................................................. 9

4 Purification ............................................................................................... 11

5 Optimization ............................................................................................ 13

6 Stripping and recharging .................................................................... 15

7 Cleaning-in-place .................................................................................. 16

8 Scaling-up ................................................................................................. 17

9 Adjusting pressure limits in chromatography system

software .................................................................................................... 18

10 Storage ...................................................................................................... 20

11 Troubleshooting ..................................................................................... 20

12 Ordering Information ........................................................................... 25

Please read these instructions carefully before using the

products.

Intended use

The products are intended for research use only, and shall not

be used in any clinical or in vitro procedures for diagnostic

purposes.

Safety

For use and handling of the products in a safe way, please refer

to the Safety Data Sheets.

2 71502768 AL

1 Product description

iTrap column characteristics

The columns are made of biocompatible polypropylene that

does not interact with biomolecules.

The columns are delivered with a stopper at the inlet and a

snap-off end at the outlet. Table 1, on page 3 lists the

characteristics of HiTrap columns.

Fig 1.

HiTrap, 1 mL column.

Fig 2.

HiTrap, 5 mL column

Note:

HiTrap columns cannot be opened or refilled.

Note:

Make sure that the connector is tight to prevent

eakage.

Table 1.

Characteristics of HiTrap columns

Column volume (CV) 1 mL 5 mL

Column dimensions 0.7 × 2.5 cm 1.6 x 2.5 cm

Column hardware pressure limit 5 bar (0.5 MPa) 5 bar (0.5 MPa)

71502768 AL 3

Note:

The pressure over the packed bed varies depending on

range of parameters such as the characteristics of

the chromatography medium, sample/liquid viscosity

and the column tubing used.

Supplied Connector kit with HiTrap column

Connectors supplied Usage No. supplied

Union 1/16” male/ luer

emale

For connection of syringe to HiTrap

column

Stop plug female, 1/16” For sealing bottom of HiTrap column 2, 5 or 7

Chromatography medium properties

isTrap HP 1 ml and 5 ml columns are prepacked with Ni

Sepharose High Performance, which consists of 34 μm highly

cross-linked agarose beads with an immobilized chelating

group. The medium has then been charged with Ni

-ions.

Several amino acids, for example histidine, form complexes

with many metal ions. Ni Sepharose High Performance

selectively binds proteins if suitable complex-forming amino

acid residues are exposed on the protein surface.

Additional histidines, such as in the case of (histidine)

-tag,

increase affinity for Ni

and generally make the histidine-

tagged protein the strongest binder among other proteins in

for example an E. coli extract.

Table 2.

HisTrap HP characteristics

Matrix Highly cross-linked spherical agarose, 6%

Average bead size 34 μm

Metal ion capacity ~ 15 μmol Ni

/ml medium

Dynamic binding capacity

At least 40 mg (histidine)

-tagged protein/ml

medium

4 71502768 AL

Recommended flow rate 1 ml/min and 5 ml/min for 1 ml and 5 ml column,

espectively

Max. flow rates

4 ml/min and 20 ml/min for 1 ml and 5 ml

column, respectively

Compatibility during use Stable in all commonly used buffers, reducing

agents, denaturants, and detergents. See Table

3, on page 6.

Chemical stability(for medium

without metal ion)

0.01 M HCl, 0.1 M NaOH; tested for one week at

40°C.

1 M NaOH, 70% acetic acid; tested for 12 h. 2%

SDS; tested for 1 h. 30% 2-propanol; tested for

30 min.

Avoid in buffers Chelating agents, e.g., EDTA, EGTA, citrate (see

able 3, on page 6)

pH stability

(for medium

without metal ion)

Working range

Cleaning-in-place

3 to 12

to 14

Storage 20% ethanol

Storage temperature 4°C to 30°C

ynamic binding capacity conditions:

Sample: 1 mg/ml (histidine)

tagged pure protein (M

28 000 or 43 000) in

binding buffer (Q

B 10%

determination) or (histidine)

-tagged protein

bound from E. coli extract

Column volume: 0.25 ml or 1 ml

Flow rate: 0.25 ml/min or 1 ml/min

Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4

Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4

Note: Dynamic binding capacity is protein-dependent.

at room temperature. For calculation of pressure limits, see Chapter 9 Adjusting

pressure limits in chromatography system software , on page 18.

Working range: pH interval where the medium can be handled without significant change in

function.

Cleaning-in-place: pH interval where the medium can be subjected to cleaning-in-place

without significant change in function.

71502768 AL 5

The Ni

-charged medium is compatible with all commonly

used aqueous buffers, reducing agents, denaturants such as 6

M Gua-HCl and 8 M urea, and a range of other additives (see

Table 3, on page 6).

Table 3. Ni Sepharose High Performance is compatible with the following

compounds up to the concentrations given

Reducing agents

5 mM DTE

mM DTT

20 mM β-mercaptoethanol

5 mM TCEP

10 mM reduced glutathione

Denaturing agents

8 M urea

M Gua-HCl

Detergents 2% Triton™ X-100 (nonionic)

% Tween™ 20 (nonionic)

2% NP-40 (nonionic)

2% cholate (anionic)

1% CHAPS (zwitterionic)

Other additives 500 mM imidazole

0% ethanol

50% glycerol

100 mM Na

1.5 M NaCl

1 mM EDTA

60 mM citrate

6 71502768 AL

Buffer 50 mM sodium phosphate, pH 7.4

00 mM Tris-HCl, pH 7.4

100 mM Tris-acetate, pH 7.4

100 mM HEPES, pH 7.4

100 mM MOPS, pH 7.4

100 mM sodium acetate, pH 4

i Sepharose High Performance is compatible with reducing agents. However, for optimal

performance, removal of any weakly bound Ni

ions by performing a blank run without

reducing agents (as described in Section Blank run, on page 10.) before applying buffer/

sample including reducing agents is recommended. Do not leave HisTrap HP columns with

buffers including reducing agents when not in use.

Tested for one week at 40°C.

The strong chelator EDTA has been used successfully in some cases at 1 mM. Generally,

chelating agents should be used with caution (and only in the sample, not in the buffers). Any

metal-ion stripping may be counteracted by addition of a small excess of MgCl

before

centrifugation/filtration of the sample. Note that stripping effects may vary with applied

sample volume.

2 General considerations

ntroduction

This section describes important information that should be

considered when using HisTrap HP in order to achieve the

best results. The actions for minimizing nickel leakage and

discoloring are normally not needed but can be performed for

sensitive applications.

Imidazole concentration

The recommended binding buffer is:

• 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM

imidazole, pH 7.4

71502768 AL 7

The imidazole concentration in sample and binding buffer can

e further increased if there is a need for higher final purity. If,

on the other hand, there is a need for higher yield the

imidazole concentration can be lowered (this may result in

lower final purity).

Minimize nickel-ion leakage

• Leakage of Ni-ions from HisTrap HP is very low under all

normal conditions. For applications where extremely low

leakage during purification is critical, leakage can be

diminished by performing a blank run. See Section Blank

run, on page 10.

• Use binding and elution buffers without reducing agents.

Reduce discoloring when reducing agents are used

HisTrap HP is compatible with reducing agents as listed in

Table 3, on page 6. Discoloring is always seen when using high

concentrations of reducing agents. In most cases this does

not affect the performance of the chromatography medium.

To minimize the discoloring, perform a blank run using buffers

without reducing agents before the purification. See Section

Blank run, on page 10.

Table 4.

Prepacked columns for desalting and buffer exchange

Column Loading volume Elution volume

HiPrep™ 26/10 Desalting

2.5 to 15 mL 7.5 to 20 mL

HiTrap Desalting

0.25 to 1.5 mL 1.0 to 2.0 mL

PD-10 Desalting

1.0 to 2.5 mL

1.75 to 2.5 mL

3.5 mL

p to 2.5 mL

PD MiniTrap™ G-25 0.1 to 2.5 mL

.2 to 0.5 mL

1.0 mL

p to 0.5 mL

8 71502768 AL

PD MidiTrap™ G-25 0.5 to 1 mL

.75 to 1 mL

1.5 mL

p to 1 mL

Prepacked with Sephadex™ G-25 Fine and requires a pump or a chromatography system to

run.

Prepacked with Sephadex G-25 Superfine and requires a syringe or pump to run.

Prepacked with Sephadex G-25 and can be run by the gravity flow or centrifugation.

Volumes with gravity elution.

Volumes with centrifugation.

3 Preparation

uffer preparation

Water and chemicals used for buffer preparation should be of

high purity. Filter buffers through a 0.22 μm or a 0.45 μm filter

before use.

Use high purity imidazole as this will give very low or no

absorbance at 280 nm.

If the recombinant histidine-tagged protein is expressed as

inclusion bodies, include 6 M Gua-HCl or 8 M urea in all buffers

and sample. On-column refolding of the denatured protein

may be possible.

Recommended conditions

Binding buffer:

20 mM sodium phosphate, 0.5 M NaCl,

0 to 40 mM imidazole, pH 7.4 (The optimal imidazole

concentration is protein dependent;

20 to 40 mM is suitable for many proteins.)

Elution buffer:

20 mM sodium phosphate, 0.5 M NaCl, 500 mM

midazole, pH 7.4

(The imidazole concentration required for elution is

protein dependent).

71502768 AL 9

leakage

Leakage of Ni

from Ni Sepharose High Performance is low

under all normal conditions. The leakage is lower than for

other IMAC media tested (see Data File 18117440). For very

critical applications, leakage during purification can be even

further diminished by performing a blank run (as described

below) before loading sample.

Blank run

Note:

Perform a blank run without reducing agents before

pplying buffers/samples containing reducing agents.

Likewise, a blank run is recommended for critical

purifications where metal ion leakage during

purification must be minimized.

Use binding buffer and elution buffer w

ithout reducing

agents.

Step Action

Wash the column with 5 column volumes (CV) of

distilled water.

Wash with 5 CV elution buffer.

Equilibrate with 10 CV binding buffer.

Note:

Ni Sepharose High Performance is compatible with

reducing agents. However, removal of any weakly

bound Ni

ions by performing a blank run without

reducing agents (as described above) before applying

buffer/ sample including reducing agents is

recommended. Do not leave HisTrap HP columns with

buffers including reducing agents when not in use.

10 71502768 AL

Sample preparation

or optimal growth, induction, and cell lysis conditions for your

recombinant histidine-tagged clones, please refer to

established protocols.

Adjust the sample to the composition and pH of the binding

buffer by:

• adding buffer, NaCl, imidazole, and additives from

concentrated stock solutions,

• diluting the sample with binding buffer, or

• buffer exchange, (see Table 4, on page 8).

Do not use strong bases or acids for pH-adjustment

(precipitation risk). Filter the sample through a 0.22 μm or a

0.45 μm filter and/or centrifuge it immediately before applying

it to the column.

To prevent the binding of host cell proteins with exposed

histidine, it is essential to include imidazole at a low

concentration in the sample and binding buffer (see Chapter 5

Optimization, on page 13).

4 Purification

Step Action

Fill the syringe or pump tubing with distilled water.

Remove the stopper and connect the column to the

yringe (use the luer connector provided), laboratory

pump or chromatography system tubing “drop-to-

drop” to avoid introducing air into the system.

71502768 AL 11

Step Action

Remove the snap-off end at the column outlet.

Wash the column with 3 to 5 column volumes of

istilled water.

Equilibrate the column with at least 5 column volumes

f binding buffer. Recommended flow rates are 1

ml/min or 5 ml/min for the 1 and 5 ml columns

respectively.

In some cases a blank run is recommended before final

equilibration/ sample application (see Section Blank

run, on page 10).

Apply the pretreated sample using a syringe or a

ump.

Wash with binding buffer until the absorbance reaches

steady baseline (generally, at least 10 to 15 column

volumes).

Note:

Purification results are improved by using imidazole in

sample and binding buffer (see Chapter 5

Optimization, on page 13).

Elute with elution buffer using a one-step or linear

radient. Five column volumes are usually sufficient if

the protein of interest is eluted by a one-step gradient.

A shallow gradient, for example a linear gradient over

20 column volumes or more, may separate proteins

with similar binding strengths.

12 71502768 AL

Note:

If imidazole needs to be removed from the protein, use

iTrap Desalting, a PD-10 Desalting Column, or HiPrep

26/10 Desalting depending on the sample volume (see

Table 4, on page 8).

5 Optimization

oncentration of imidazole

Imidazole at low concentrations is commonly used in the

binding and the wash buffers to minimize binding of host cell

proteins. For the same reason, it is important to also include

imidazole in the sample (generally, at the same concentration

as in the wash buffer). At somewhat higher concentrations,

imidazole may also decrease the binding of histidine-tagged

proteins.

The imidazole concentration must therefore be optimized to

ensure the best balance of high purity (low binding of host cell

proteins), and high yield (binding of histidine-tagged target

protein). This optimal concentration is different for different

histidine-tagged proteins, and is usually slightly higher for Ni

Sepharose High Performance than for similar IMAC media on

the market (see Data File 18117440).

Finding the optimal imidazole concentration for a specific

histidine-tagged protein is a trial-and-error effort, but 20 to 40

mM in the binding and wash buffers is a good starting point for

many proteins. Use a high purity imidazole, such imidazole

gives essentially no absorbance at 280 nm.

71502768 AL 13

Choice of metal ion

is usually the first choice metal ion for purifying most

(histidine)

-tagged recombinant proteins from nontagged

host cell proteins, and also the ion most generally used.

Nevertheless, it is not always possible to predict which metal

ion will be best for a given protein. The strength of binding

between a protein and a metal ion is affected by several

factors, including the length, position, and exposure of the

affinity tag on the protein, the type of ion used, and the pH of

buffers, so some proteins may be easier to purify with ions

other than Ni

A quick and efficient way to test this possibility and optimize

separation conditions is to use HiTrap IMAC HP 1 ml columns,

which are packed with IMAC Sepharose High Performance

(not charged with metal ions). Each column can be charged

with different metal ions, for example Cu

, Co

, Zn

, Ca

, or

. Instructions are included with each column.

A study to compare the purification of six (histidine)

-tagged

recombinant proteins, including three variants of maltose-

binding protein, with different metal ions has indicated that

generally gives best selectivity between (histidine)-

tagged and nontagged host-cell proteins (see Application

Note 18114518).

14 71502768 AL

6 Stripping and recharging

Note:

The column does not have to be stripped and

echarged between each purification if the same

protein is going to be purified; it is sufficient to strip

and recharge it after five to seven purifications,

depending on the cell extract, extract volume, target

protein, etc.

Stripping

Recommended stripping buffer: 20 mM sodium

phosphate, 0.5 M NaCl, 50 mM EDTA, pH 7.4

Strip the column by washing with at least 5 to 10 column

volumes of stripping buffer. Wash with at least 5 to 10 column

volumes of binding buffer and 5 to 10 column volumes of

distilled water before recharging the column.

Recharging

Recharge the water-washed column by loading 0.5 ml or 2.5

ml of 0.1 M NiSO

in distilled water on HisTrap HP 1 ml and 5 ml

column, respectively. Salts of other metals, chlorides, or

sulfates, may also be used (see Chapter 5 Optimization, on

page 13).

Wash with 5 column volumes distilled water, and 5 column

volumes binding buffer (to adjust pH) before storage in 20%

ethanol.

71502768 AL 15

7 Cleaning-in-place

hen an increase in back pressure is seen, the column should

be cleaned. Before cleaning, strip off Ni

ions using the

recommended procedure described in Chapter 6 Stripping

and recharging, on page 15.

After cleaning, store in 20% ethanol (wash with 5 column

volumes) or recharge with Ni

prior to storage in ethanol.

The Ni

-stripped column can be cleaned by the following

Cleaning-in-place (CIP) protocols:

CIP protocols

Ionically bound proteins Wash with several column volumes of 1.5 M

aCl; then wash with approx. 10 column

volumes of distilled water.

Precipitated proteins,

hydrophobically bound proteins,

and lipoproteins

Wash the column with 1 M NaOH, contact

time usually 1 to 2 hours (12 hours or more

for endotoxin removal). Then wash with

approx. 10 column volumes of binding

buffer, followed by 5 to 10 column volumes

of distilled water.

16 71502768 AL

Hydrophobically bound proteins,

ipoproteins, and lipids

Wash with 5 to 10 column volumes of 30%

iso-propanol for about 15 to 20 minutes.

Then wash with approx. 10 column volumes

of distilled water.

Alternatively, wash with 2 column volumes

of detergent in a basic or acidic solution.

Use, for example, 0.1 to 0.5% nonionic

detergent in 0.1 M acetic acid, contact time

1 to 2 hours. After treatment, always

remove residual detergent by washing with

at least 5 column volumes of 70% ethanol

Then wash with approx. 10 column volumes

of distilled water.

pecific regulations may apply when using 70% ethanol since the use of explosion proof

areas and equipment may be required.

8 Scaling-up

wo or three HisTrap HP 1 ml or 5 ml columns can be

connected in series for quick scale-up (note that back-

pressure will increase).

Ni Sepharose High Performance, the medium prepacked in

HisTrap HP columns, is supplied preswollen in 25 and 100 ml

lab packs (see Chapter 12 Ordering Information, on page 25).

An alternative scale-up strategy is thus to pack the medium in

empty columns – Tricorn™ and XK columns are suitable for

this purpose.

71502768 AL 17

9 Adjusting pressure limits in

hromatography system software

Pressure generated by the flow through a column affects the

packed bed and the column hardware, see Figure 3, on page

18 below. Increased pressure is generated when running/

using one or a combination of the following conditions:

• High flow rates

• Buffers or sample with high viscosity

• Low temperature

• A flow restrictor

Note:

Exceeding the flow limit (see recommended flow rates

n Table 2, on page 4) can damage the column.

pre-column pressure

post-column pressure

pressure over the

packed bed

Fig 3.

Precolumn and post-column measurements.

18 71502768 AL

ÄKTA avant and ÄKTA pure

he system will automatically monitor the pressures (pre-

column pressure and pressure over the packed bed, Δp). The

pre-column pressure limit is the column hardware pressure

limit (see Table 1, on page 3).

The maximum pressure the packed bed can withstand

depends on media characteristics and sample/liquid viscosity.

The measured value also depends on the tubing used to

connect the column to the instrument.

ÄKTAexplorer, ÄKTApurifier, ÄKTAFPLC and other

systems with pressure sensor in the pump

To obtain optimal functionality, the pressure limit in the

software may be adjusted according to the following

procedure:

Step Action

Replace the column with a piece of tubing. Run the

ump at the maximum intended flow rate. Note the

pressure as total system pressure, P1.

Disconnect the tubing and run the pump at the same

low rate used in step 1. Note that there will be a drip

from the column valve. Note this pressure as P2.

71502768 AL 19

Step Action

Calculate the new pressure limit as a sum of P2 and the

olumn hardware pressure limit (see Table 1, on page

3). Replace the pressure limit in the software with the

calculated value.

The actual pressure over the packed bed (Δp) will

during run be equal to actual measured pressure -

total system pressure (P1).

Note:

Repeat the procedure each time the parameters are

hanged.

10 Storage

tore HisTrap HP columns in 20% ethanol at 4°C to 30°C.

11 Troubleshooting

The following tips may be of assistance. If you have any further

questions about HisTrap HP, please visit cytiva.com, contact

our technical support, or your local Cytiva representative.

Note:

When using high concentrations of urea or Gua-HCl,

rotein unfolding generally takes place. Refolding

oncolumn (or after elution) is protein dependent.

Tip:

To minimize dilution of the sample, solid urea or Gua-

Cl can be added.

20 71502768 AL

Tip:

Samples containing urea can be analyzed directly by

DS-PAGE whereas samples containing Gua-HCl must

be buffer-exchanged to a buffer with urea before SDS-

PAGE.

Column has clogged

• C

ell debris in the sample may clog the column. Clean the

column according to Chapter 7 Cleaning-in-place, on page

• Centrifuge and/or filter the sample through a 0.22 μm or a

0.45 μm filter, see Sample preparation, on page 11.

Sample is too viscous

• If the lysate is very viscous due to high concentration of

host nucleic acid, continue sonication until the viscosity is

reduced, and/or add DNase I to 5 μg/ml, Mg

to 1 mM, and

incubate on ice for 10 to 15 minutes. Alternatively, draw

the lysate through a syringe needle several times.

Protein is difficult to dissolve or precipitates during

purification

• See Table 3, on page 6 for reducing agents,

detergents, glycerol and denaturing agents that may

be used.

Mix gently for 30 minutes after addition of additives to aid

solubilization of the tagged protein (inclusion bodies may

require much longer mixing). Note that Triton X-100 and

NP-40 (but not Tween) have a high absorbance at 280 nm.

Furthermore, detergents cannot be easily removed by

buffer exchange.

71502768 AL 21

No histidine-tagged protein in the purified fractions

• E

lution conditions are too mild (histidine-tagged

protein still bound): Elute with an increasing imidazole

gradient or decreasing pH to determine the optimal

elution conditions.

• The protein has precipitated in the column: For the

next experiment, decrease amount of sample, or decrease

protein concentration by eluting with linear imidazole

gradient instead of imidazole steps. Try detergents or

changed NaCl concentration, or elute under denaturing

(unfolding) conditions (add 4 to 8 M urea or 4 to 6 M Gua-

HCl).

• Nonspecific hydrophobic or other interaction: Add a

nonionic detergent to the elution buffer (e.g., 0.2% Triton

X-100) or increase the NaCl concentration.

• Concentration of imidazole in the sample and/or

binding buffer is too high: The protein is found in the

flowthrough material. Decrease the imidazole

concentration.

• Histidine-tag may be insufficiently exposed: The

protein is found in the flowthrough material; perform

purification of unfolded protein in urea or Gua-HCl as for

inclusion bodies.

• Buffer/sample composition is incorrect: The protein is

found in the flowthrough material. Check pH and

composition of sample and binding buffer. Make sure that

chelating or strong reducing agents are not present in the

sample at too high concentration, and that the

concentration of imidazole is not too high.

22 71502768 AL

SDS-PAGE of samples collected during the preparation of the

acterial lysate may indicate that most of histidine-tagged

protein is located in the centrifugation pellet. Possible causes

and solutions are:

• Sonication may be insufficient: Cell disruption may be

checked by microscopic examination or monitored by

measuring the release of nucleic acids at A

260

. Addition of

lysozyme (up to

0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-

HCl, pH 8.0) prior to sonication may improve results. Avoid

frothing and overheating as this may denature the target

protein. Over-sonication can also lead to copurification of

host proteins with the target protein.

• The protein may be insoluble (inclusion bodies): The

protein can usually be solubilized (and unfolded) from

inclusion bodies using common denaturants such as 4 to

6 M Gua-HCl, 4 to 8 M urea, or strong detergents. Prepare

buffers containing 20 mM sodium phosphate, 8 M urea, or

6 M Gua-HCl, and suitable imidazole concentrations, pH

7.4 to 7.6. Buffers with urea should also include 500 mM

NaCl. Use these buffers for sample preparation, as binding

buffer and as elution buffer. For sample preparation and

binding buffer, use 10 to 20 mM imidazole or the

concentration selected during optimization trials

(including urea or Gua-HCl).

The eluted protein is not pure (multiple bands on SDS

polyacrylamide gel)

• Partial degradation of tagged protein by proteases:

Add protease inhibitors (use EDTA with caution, see Table

3, on page 6).

71502768 AL 23

• C

ontaminants have high affinity for nickel ions: Elute

with a stepwise or linear imidazole gradient to determine

optimal imidazole concentrations to use for binding and

for wash; add imidazole to the sample in the same

concentration as the binding buffer. Wash before elution

with binding buffer containing as high concentration of

imidazole as possible, without causing elution of the

tagged protein. A shallow imidazole gradient (20 column

volumes or more), may separate proteins with similar

binding strengths. If optimized conditions do not remove

contaminants, further purification by ion exchange

chromatography (HiTrap Q HP or HiTrap SP HP) and/or gel

filtration (Superdex™ Peptide, Superdex 75 or Superdex

200) may be necessary.

• Contaminants are associated with tagged proteins:

Add detergent and/or reducing agents before sonicating

cells. Increase detergent levels (e.g. up to 2% Triton X-100

or 2% Tween 20), or add glycerol (up to 50%) to the wash

buffer to disrupt nonspecific interactions.

Histidine-tagged protein is eluted during sample

loading/wash

• Buffer/sample composition is incorrect: Check pH and

composition of sample and binding buffer. Ensure that

chelating or strong reducing agents are not present in the

sample at a too high concentration, and that the

concentration of imidazole is not too high.

• Histidine-tag is partially obstructed: Purify under

denaturing conditions (use 4 to 8 M urea or 4 to 6 M Gua-

HCl).

24 71502768 AL

• C

olumn capacity is exceeded: Join two or three

HisTrap HP 1 ml columns together or change to a HisTrap

HP 5 ml column.

12 Ordering Information

Product QuantityProduct Code.

HisTrap HP 1 × 1 ml 29051021

5 × 1 ml 17524701

100 × 1 ml

17524705

1 × 5 ml 17524801

5 × 5 ml 17524802

100 × 5 ml

17524805

ack size available by special order.