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2024年4月13日发(作者：慈晨欣)

Instruction Manual

ProBond

Purification System

For purification of polyhistidine-containing

recombinant proteins

Catalog nos. K850-01, K851-01, K852-01, K853-01, K854-01,

R801-01, R801-15

Version K

2 September2004

25-0006

Table of Contents

Kit Contents iv

Preparing 2

Purification Procedure—.7

Purification Procedure—11

Purification Procedure—13

Frequently 21

iii

Kit Contents and Storage

Types of Products

This manual is supplied with the following products:

Product Catalog No.

ProBond

™

Purification System

Components

ProBond

™

Purification System K850-01

ProBond

™

Purification System with Antibody

with Anti-Xpress

™

Antibody

K851-01

with Anti-myc-HRP Antibody

K852-01

with Anti-His(C-term)-HRP Antibody

K853-01

with Anti-V5-HRP Antibody

K854-01

ProBond

™

Nickel-Chelating Resin (50 ml) R801-01

ProBond

™

Nickel Chelating Resin (150 ml) R801-15

The ProBond

™

Purification System includes enough resin, reagents, and

columns for six purifications. The components are listed below. See next page

for resin specifications.

Component Composition Quantity

ProBond

™

Resin 50% slurry in 20% ethanol 12 ml

5X Native 250 mM NaH

, pH 8.0

1 × 125 ml bottle

Purification Buffer

2.5 M NaCl

Guanidinium Lysis 6 M Guanidine HCl

1 × 60 ml bottle

Buffer

20 mM sodium phosphate, pH 7.8

500 mM NaCl

Denaturing 8 M Urea

Binding Buffer

2 × 125 ml bottles

20 mM sodium phosphate, pH 7.8

500 mM NaCl

Denaturing Wash 8 M Urea

2 × 125 ml bottles

Buffer

20 mM sodium phosphate, pH 6.0

500 mM NaCl

Denaturing Elution 8 M Urea

1 × 60 ml bottle

Buffer

20 mM NaH

, pH 4.0

500 mM NaCl

Imidazole 3 M Imidazole,

1 × 8 ml bottle

20 mM sodium phosphate, pH 6.0

500 mM NaCl

Purification 10 ml columns 6

Columns

Continued on next page

Kit Contents and Storage,

Continued

The ProBond

™

Purification System with Antibody includes resin, reagents, and

ProBond

Purification System

columns as described for the ProBond

™

Purification System (previous page)

™

with Antibody

Storage

Resin and Column

Specifications

Product

Qualification

and 50 µl of the appropriate purified mouse monoclonal antibody. Sufficient

reagents are included to perform six purifications and 25 Western blots with the

antibody.

For more details on the antibody specificity, subclass, and protocols for using

the antibody, refer to the antibody manual supplied with the system.

Store ProBond

™

resin at +4°C. Store buffer and columns at room temperature.

Store the antibody at 4°C. Avoid repeated freezing and thawing of the

antibody as it may result in loss of activity.

The product is guaranteed for 6 months when stored properly.

All native purification buffers are prepared from the 5X Native Purification

Buffer and the 3 M Imidazole, as described on page 7.

The Denaturing Wash Buffer pH 5.3 is prepared from the Denaturing Wash

Buffer (pH 6.0), as described on page 11.

ProBond

™

resin is precharged with Ni

ions and appears blue in color. It is

provided as a 50% slurry in 20% ethanol.

ProBond

™

resin and purification columns have the following specifications:

• Binding capacity of ProBond

™

resin: 1–5 mg of protein per ml of resin

• Average bead size: 45–165 microns

• Pore size of purification columns: 30–35 microns

• Recommended flow rate: 0.5 ml/min

• Maximum flow rate: 2 ml/min

• Maximum linear flow rate: 700 cm/h

• Column material: Polypropylene

• pH stability (long term): pH 3–13

• pH stability (short term): pH 2–14

The ProBond

™

Purification System is qualified by purifying 2 mg of myoglobin

protein on a column and performing a Bradford assay. Protein recovery must

be 75% or higher.

Accessory Products

Additional

Products

Pre-Cast Gels and

Pre-made Buffers

The following products are also available for order from Invitrogen:

Product Quantity Catalog No.

ProBond

™

Nickel-Chelating 50 ml R801-01

Resin

150 ml R801-15

Polypropylene columns 50 R640-50

(empty)

Ni-NTA Agarose 10 ml R901-01

25 ml R901-15

Ni-NTA Purification System 6 purifications K950-01

Ni-NTA Purification System

with Antibody

with Anti-Xpress

™

Antibody

1 kit

K951-01

with Anti-myc-HRP Antibody

1 kit

K952-01

with Anti-His(C-term)-HRP

1 kit

K953-01

Antibody

with Anti-V5-HRP Antibody

1 kit K954-01

Anti-myc Antibody

50 µl

R950-25

Anti-V5 Antibody

50 µl

R960-25

Anti-Xpress

™

Antibody

50 µl

R910-25

Anti-His(C-term) Antibody

50 µl

R930-25

InVision

™

His-tag In-gel Stain 500 ml LC6030

InVision

™

His-tag In-gel 1 kit LC6033

Staining Kit

A large variety of pre-cast gels for SDS-PAGE and pre-made buffers for your

convenience are available from Invitrogen. For details, visit our web site at

or contact Technical Service (page 23).

Introduction

Overview

Introduction

ProBond

™

Nickel-

Chelating Resin

Binding

Characteristics

Native Versus

Denaturing

Conditions

The ProBond

™

Purification System is designed for purification of 6xHis-tagged

recombinant proteins expressed in bacteria, insect, and mammalian cells. The

system is designed around the high affinity and selectivity of ProBond

™

Nickel-Chelating Resin for recombinant fusion proteins containing six tandem

histidine residues.

The ProBond

™

Purification System is a complete system that includes

purification buffers and resin for purifying proteins under native, denaturing,

or hybrid conditions. The resulting proteins are ready for use in many target

applications.

This manual is designed to provide generic protocols that can be adapted for

your particular proteins. The optimal purification parameters will vary with

each protein being purified.

ProBond

™

Nickel-Chelating Resin is used for purification of recombinant

proteins expressed in bacteria, insect, and mammalian cells from any 6xHis-

tagged vector. ProBond

™

Nickel-Chelating Resin exhibits high affinity and

selectivity for 6xHis-tagged recombinant fusion proteins.

Proteins can be purified under native, denaturing, or hybrid conditions using

the ProBond

™

Nickel-Chelating Resin. Proteins bound to the resin are eluted

with low pH buffer or by competition with imidazole or histidine. The resulting

proteins are ready for use in target applications.

ProBond

™

Nickel-Chelating Resin uses the chelating ligand iminodiacetic acid

(IDA) in a highly cross-linked agarose matrix. IDA binds Ni

ions by three

coordination sites.

The protocols provided in this manual are generic, and may not result in 100%

pure protein. These protocols should be optimized based on the binding

characteristics of your particular proteins.

The decision to purify your 6xHis-tagged fusion proteins under native or

denaturing conditions depends on the solubility of the protein and the need to

retain biological activity for downstream applications.

• Use native conditions if your protein is soluble (in the supernatant after

lysis) and you want to preserve protein activity.

• Use denaturing conditions if the protein is insoluble (in the pellet after

lysis) or if your downstream application does not depend on protein

activity.

•

Use hybrid protocol if your protein is insoluble but you want to preserve

protein activity. Using this protocol, you prepare the lysate and columns

under denaturing conditions and then use native buffers during the wash

and elution steps to refold the protein. Note that this protocol may not

restore activity for all proteins. See page 14.

Methods

Preparing Cell Lysates

Introduction

Materials Needed

Processing Higher

Amount of Starting

Material

Instructions for preparing lysates from bacteria, insect, and mammalian cells

using native or denaturing conditions are described below.

You will need the following items:

• Native Binding Buffer (recipe is on page 8) for preparing lysates under

native conditions

• Sonicator

• 10 µg/ml RNase and 5 µg/ml DNase I (optional)

• Guanidinium Lysis Buffer (supplied with the system) for preparing lysates

under denaturing conditions

• 18-gauge needle

• Centrifuge

• Sterile, distilled water

• SDS-PAGE sample buffer

• Lysozyme for preparing bacterial cell lysates

• Bestatin or Leupeptin, for preparing mammalian cell lysates

Instructions for preparing lysates from specific amount of starting material

(bacteria, insect, and mammalian cells) and purification with 2 ml resin under

native or denaturing conditions are described in this manual.

If you wish to purify your protein of interest from higher amounts of starting

material, you may need to optimize the lysis protocol and purification

conditions (amount of resin used for binding). The optimization depends on the

expected yield of your protein and amount of resin to use for purification.

Perform a pilot experiment to optimize the purification conditions and then

based on the pilot experiment results, scale-up accordingly.

Continued on next page

Preparing Cell Lysates

, Continued

Preparing Bacterial

Follow the procedure below to prepare bacterial cell lysate under native

Cell Lysate—Native

conditions. Scale up or down as necessary.

Conditions

1. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for

5 minutes in a Sorvall SS-34 rotor). Resuspend the cells in 8 ml Native

Binding Buffer (recipe on page 8).

Add 8 mg lysozyme and incubate on ice for 30 minutes.

Using a sonicator equipped with a microtip, sonicate the solution on ice

using six 10-second bursts at high intensity with a 10-second cooling

period between each burst.

Alternatively, sonicate the solution on ice using two or three 10-second

bursts at medium intensity, then flash freeze the lysate in liquid nitrogen

or a methanol dry ice slurry. Quickly thaw the lysate at 37°C and

perform two more rapid sonicate-freeze-thaw cycles.

Optional: If the lysate is very viscous, add RNase A (10 µg/ml) and

DNase I (5 µg/ml) and incubate on ice for 10–15 minutes. Alternatively,

draw the lysate through a 18-gauge syringe needle several times.

Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular

debris. Transfer the supernatant to a fresh tube.

Note: Some 6xHis-tagged protein may remain insoluble in the pellet, and

can be recovered by preparing a denatured lysate (page 4) followed by

the denaturing purification protocol (page 12). To recover this insoluble

protein while preserving its biological activity, you can prepare the

denatured lysate and then follow the hybrid protocol on page 14. Note

that the hybrid protocol may not restore activity in all cases, and should

be tested with your particular protein.

6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaining

lysate on ice or freeze at -20°C. When ready to use, proceed to the

protocol on page 7.

Continued on next page

Preparing Cell Lysates

, Continued

Preparing Bacterial

Follow the procedure below to prepare bacterial cell lysate under denaturing

Cell Lysate—

Denaturing

Conditions

Harvesting Insect

Cells

conditions:

1. Equilibrate the Guanidinium Lysis Buffer, pH 7.8 (supplied with the

system or see page 19 for recipe) to 37°C.

2. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for

5 minutes in a Sorvall SS-34 rotor).

3. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer from Step 1.

4. Slowly rock the cells for 5–10 minutes at room temperature to ensure

thorough cell lysis.

5. Sonicate the cell lysate on ice with three 5-second pulses at high intensity.

6. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular

debris.

Transfer the supernatant to a fresh tube.

7. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaining

lysate on ice or at -20°C. When ready to use, proceed to the denaturing

protocol on page 11 or hybrid protocol on page 13.

Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer,

you need to dilute the samples, dialyze the samples, or perform TCA

precipitation prior to SDS-PAGE to prevent the precipitation of SDS.

For detailed protocols dealing with insect cell expression, consult the manual

for your particular system. The following lysate protocols are for baculovirus-

infected cells and are intended to be highly generic. They should be optimized

for your cell lines.

For baculovirus-infected insect cells, when the time point of maximal

expression has been determined, large scale protein expression can be carried

out. Generally, the large-scale expression is performed in 1 liter flasks seeded

with cells at a density of 2 × 10

cells/ml in a total volume of 500 ml and

infected with high titer viral stock at an MOI of 10 pfu/cell. At the point of

maximal expression, harvest cells in 50 ml aliquots. Pellet the cells by

centrifugation and store at -70°C until needed. Proceed to preparing cell lysates

using native or denaturing conditions as described on the next page.

Continued on next page

Preparing Cell Lysates

, Continued

Preparing Insect

Cell Lysate—Native

1. Prepare 8 ml Native Binding Buffer (recipe on page 8) containing

Leupeptin (a protease inhibitor) at a concentration of 0.5 µg/ml.

Condition

Preparing Insect

Cell Lysate—

Denaturing

Condition

2. After harvesting the cells (previous page), resuspend the cell pellet in

8 ml Native Binding Buffer containing 0.5 µg/ml Leupeptin.

3. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dry

ice/ethanol bath and a 42°C water bath.

4. Shear DNA by passing the preparation through an 18-gauge needle four

times.

5. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular

debris.

Transfer the supernatant to a fresh tube.

6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysate

on ice or freeze at -20°C. When ready to use, proceed to the protocol on

page 7.

1. After harvesting insect cells (previous page), resuspend the cell pellet in

8 ml Guanidinium Lysis Buffer (supplied with the system or see page 19

for recipe).

2. Pass the preparation through an 18-gauge needle four times.

3. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular

debris. Transfer the supernatant to a fresh tube.

4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysate

on ice or freeze at -20° C. When ready to use, proceed to the denaturing

protocol on page 11 or hybrid protocol on page 13.

Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer,

you need to dilute the samples, dialyze the samples, or perform TCA

precipitation prior to SDS-PAGE to prevent the precipitation of SDS.

Continued on next page

Preparing Cell Lysates

, Continued

Preparing

Mammalian Cell

Lysate—Native

Conditions

Preparing

Mammalian Cell

Lysates—

Denaturing

Conditions

For detailed protocols dealing with mammalian expression, consult the manual

for your particular system. The following protocols are intended to be highly

generic, and should be optimized for your cell lines.

To produce recombinant protein, you need between 5 x 10

and 1 x 10

cells.

Seed cells and grow in the appropriate medium until they are 80–90%

confluent. Harvest cells by trypsinization. You can freeze the cell pellet in

liquid nitrogen and store at -70°C until use.

1. Resuspend the cell pellet in 8 ml of Native Binding Buffer (page 8). The

addition of protease inhibitors such as bestatin and leupeptin may be

necessary depending on the cell line and expressed protein.

2. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dry

ice/ethanol bath and a 42°C water bath.

3. Shear the DNA by passing the preparation through an 18-gauge needle

four times.

4. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular

debris. Transfer the supernatant to a fresh tube.

5. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaining

lysate on ice or freeze at -20° C. When ready to use, proceed to the

protocol on page 7.

For detailed protocols dealing with mammalian expression, consult the manual

for your particular system. The following protocols are intended to be highly

generic, and should be optimized for your cell lines.

To produce recombinant protein, you need between 5 x 10

and 1 x 10

cells.

Seed cells and grow in the appropriate medium until they are 80–90%

confluent. Harvest cells by trypsinization. You can freeze the cell pellet in

liquid nitrogen and store at -70°C until use.

1. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer (supplied

with the system or see page 19 for recipe).

2. Shear the DNA by passing the preparation through an 18-gauge needle

four times.

3. Centrifuge the lysate at 3,000 × g for 15 minutes to pellet the cellular

debris. Transfer the supernatant to a fresh tube.

4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaining

lysate on ice or freeze at -20° C until use. When ready to use, proceed to

the denaturing protocol on page 11 or hybrid protocol on page 13.

Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer,

you need to dilute the samples, dialyze the samples, or perform TCA

precipitation prior to SDS-PAGE to prevent the precipitation of SDS.

Purification Procedure—Native Conditions

Introduction

Buffers for Native

Purification

Materials Needed

Imidazole

Concentration in

Native Buffers

In the following procedure, use the prepared Native Binding Buffer, Native

Wash Buffer, and Native Elution Buffer, columns, and cell lysate prepared

under native conditions. Be sure to check the pH of your buffers before starting.

All buffers for purification under native conditions are prepared from the

5X Native Purification Buffer supplied with the system. Dilute and adjust the

pH of the 5X Native Purification Buffer to create 1X Native Purification Buffer

(page 8). From this, you can create the following buffers:

• Native Binding Buffer

• Native Wash Buffer

• Native Elution Buffer

The recipes described in this section will create sufficient buffers to perform one

native purification using one kit-supplied purification column. Scale up

accordingly.

If you are preparing your own buffers, see page 18 for recipe.

You will need the following items:

• 5X Native Purification Buffer (supplied with the system or see page 18 for

recipe)

• 3 M Imidazole (supplied with the system or see page 18 for recipe)

• NaOH

• HCl

• Sterile distilled water

• Prepared ProBond

™

columns with native buffers (next page)

• Lysate prepared under native conditions (page 2)

Imidazole is included in the Native Wash and Elution Buffers to minimize the

binding of untagged, contaminating proteins and increase the purity of the

target protein with fewer wash steps. Note that, if your level of contaminating

proteins is high, you may add imidazole to the Native Binding Buffer.

If your protein does not bind well under these conditions, you can experiment

with lowering or eliminating the imidazole in the buffers and increasing the

number of wash and elution steps.

Continued on next page

Purification Procedure—Native Conditions

, Continued

1X Native

Purification Buffer

Native Binding

Buffer

Native Wash Buffer

Native Elution

Buffer

To prepare 100 ml 1X Native Purification Buffer, combine:

• 80 ml of sterile distilled water

• 20 ml of 5X Native Purification Buffer (supplied with the system or see

page 18 for recipe)

Mix well and adjust pH to 8.0 with NaOH or HCl.

Without Imidazole

Use 30 ml of the 1X Native Purification Buffer (see above for recipe) for use as

the Native Binding Buffer (used for column preparation, cell lysis, and

binding).

With Imidazole (Optional):

You can prepare the Native Binding Buffer with imidazole to reduce the

binding of contaminating proteins. (Note that some His-tagged proteins may

not bind under these conditions.).

To prepare 30 ml Native Binding Buffer with 10 mM imidazole, combine:

• 30 ml of 1X Native Purification Buffer

• 100 µl of 3 M Imidazole, pH 6.0

Mix well and adjust pH to 8.0 with NaOH or HCl.

To prepare 50 ml Native Wash Buffer with 20 mM imidazole, combine:

• 50 ml of 1X Native Purification Buffer

• 335 µl of 3 M Imidazole, pH 6.0

Mix well and adjust pH to 8.0 with NaOH or HCl.

To prepare 15 ml Native Elution Buffer with 250 mM imidazole, combine:

• 13.75 ml of 1X Native Purification Buffer

• 1.25 ml of 3 M Imidazole, pH 6.0

Mix well and adjust pH to 8.0 with NaOH or HCl.

Continued on next page

Purification Procedure—Native Conditions

, Continued

Preparing

ProBond

™

Column

Storing Prepared

Columns

Do not use strong reducing agents such as DTT with ProBond

™

columns. DTT

reduces the nickel ions in the resin. In addition, do not use strong chelating

agents such as EDTA or EGTA in the loading buffers or wash buffers, as these

will strip the nickel from the columns.

Be sure to check the pH of your buffers before starting.

When preparing a column as described below, make sure that the snap-off cap

at the bottom of the column remains intact. To prepare a column:

1. Resuspend the ProBond

™

resin in its bottle by inverting and gently

tapping the bottle repeatedly.

2. Pipet or pour 2 ml of the resin into a 10-ml Purification Column

supplied with the kit. Allow the resin to settle completely by gravity

(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minute

at 800 × g). Gently aspirate the supernatant.

3. Add 6 ml of sterile, distilled water and resuspend the resin by

alternately inverting and gently tapping the column.

4. Allow the resin to settle using gravity or centrifugation as described in

Step 2, and gently aspirate the supernatant.

5. For purification under Native Conditions, add 6 ml Native Binding

Buffer (recipe on page 8).

6. Resuspend the resin by alternately inverting and gently tapping the

column.

7. Allow the resin to settle using gravity or centrifugation as described in

Step 2, and gently aspirate the supernatant.

8. Repeat Steps 5 through 7.

To store a column containing resin, add 0.02% azide or 20% ethanol as a

preservative and cap or parafilm the column. Store at room temperature.

Continued on next page

Purification Procedure—Native Conditions

, Continued

Purification Under

Native Conditions

Using the native buffers, columns and cell lysate, follow the procedure below to

purify proteins under native conditions:

1. Add 8 ml of lysate prepared under native conditions to a prepared

Purification Column (page 9).

2. Bind for 30–60 minutes using gentle agitation to keep the resin

suspended in the lysate solution.

3. Settle the resin by gravity or low speed centrifugation (800 × g), and

carefully aspirate the supernatant. Save supernatant at 4°C for

SDS-PAGE analysis.

4. Wash with 8 ml Native Wash Buffer (page 8). Settle the resin by gravity

or low speed centrifugation (800 × g), and carefully aspirate the

supernatant. Save supernatant at 4°C for SDS-PAGE analysis.

5. Repeat Step 4 three more times.

6. Clamp the column in a vertical position and snap off the cap on the

lower end. Elute the protein with 8–12 ml Native Elution Buffer (see

page 2). Collect 1 ml fractions and analyze with SDS-PAGE.

Note: Store the eluted fractions at 4°C. If -20°C storage is required, add

glycerol to the fractions. For long term storage, add protease inhibitors to

the fractions.

If you wish to reuse the resin to purify the same recombinant protein, wash the

resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable

binding buffer. If you need to recharge the resin, see page 17.

Purification Procedure—Denaturing Conditions

Introduction

Materials Needed

Preparing the

Denaturing Wash

Buffer pH 5.3

Preparing

ProBond

™

Column

Instructions to perform purification using denaturing conditions with prepared

denaturing buffers, columns, and cell lysate are described below.

You will need the following items:

• Denaturing Binding Buffer (supplied with the system or see page 19 for

recipe)

• Denaturing Wash Buffer, pH 6.0 (supplied with the system or see page 19 for

recipe) and Denaturing Wash Buffer, pH 5.3 (see recipe below)

• Denaturing Elution Buffer (supplied with the system or see page 20 for

recipe)

• Prepared ProBond

™

columns with Denaturing buffers (see below)

• Lysate prepared under denaturing conditions (page 11)

Using a 10 ml aliquot of the kit-supplied Denaturing Wash Buffer (pH 6.0), mix

well, and adjust the pH to 5.3 using HCl. Use this for the Denaturing Wash

Buffer pH 5.3 in Step 5 next page.

Be sure to check the pH of your buffers before starting. Note that the

denaturing buffers containing urea will become more basic over time.

When preparing a column as described below, make sure that the snap-off cap

at the bottom of the column remains intact.

If you are reusing the ProBond

™

resin, see page 17 for recharging protocol.

To prepare a column:

1. Resuspend the ProBond

™

resin in its bottle by inverting and gently

tapping the bottle repeatedly.

2. Pipet or pour 2 ml of the resin into a 10-ml Purification Column

supplied with the kit. Allow the resin to settle completely by gravity

(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minute

at 800 × g). Gently aspirate the supernatant.

3. Add 6 ml of sterile, distilled water and resuspend the resin by

alternately inverting and gently tapping the column.

4. Allow the resin to settle using gravity or centrifugation as described in

Step 2, and gently aspirate the supernatant.

5. For purification under Denaturing Conditions, add 6 ml of Denaturing

Binding Buffer.

6. Resuspend the resin by alternately inverting and gently tapping the

column.

7. Allow the resin to settle using gravity or centrifugation as described in

Step 2, and gently aspirate the supernatant. Repeat Steps 5 through 7.

Continued on next page

Purification Procedure—Denaturing Conditions,

Continued

Purification Under

Denaturing

Conditions

Using the denaturing buffers, columns, and cell lysate, follow the procedure

below to purify proteins under denaturing conditions:

1. Add 8 ml lysate prepared under denaturing conditions to a prepared

Purification Column (page 11).

2. Bind for 15–30 minutes at room temperature using gentle agitation (e.g.,

using a rotating wheel) to keep the resin suspended in the lysate

solution. Settle the resin by gravity or low speed centrifugation (800 × g),

and carefully aspirate the supernatant.

3. Wash the column with 4 ml Denaturing Binding Buffer supplied with the

kit by resuspending the resin and rocking for two minutes. Settle the

resin by gravity or low speed centrifugation (800 × g), and carefully

aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE

analysis. Repeat this step one more time.

4. Wash the column with 4 ml Denaturing Wash Buffer, pH 6.0 supplied in

the kit by resuspending the resin and rocking for two minutes. Settle the

resin by gravity or low speed centrifugation (800 × g), and carefully

aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE

analysis. Repeat this step one more time.

5. Wash the column with 4 ml Denaturing Wash Buffer pH 5.3 (see recipe

on previous page) by resuspending the resin and rocking for 2 minutes.

Settle the resin by gravity or low speed centrifugation (800 × g), and

carefully aspirate the supernatant. Save supernatant at 4°C for SDS-

PAGE analysis. Repeat this step once more for a total of two washes with

Denaturing Wash Buffer pH 5.3.

6. Clamp the column in a vertical position and snap off the cap on the

lower end. Elute the protein by adding 5 ml Denaturing Elution Buffer

supplied with the kit. Collect 1 ml fractions and monitor the elution by

taking OD

280

readings of the fractions. Pool the fractions that contain the

peak absorbance and dialyze against 10 mM Tris, pH 8.0, 0.1% Triton X-

100 overnight at 4°C to remove the urea. Concentrate the dialyzed

material by any standard method (i.e., using 10,000 MW cut-off, low-

protein binding centrifugal instruments or vacuum concentration

instruments).

If you wish to reuse the resin to purify the same recombinant protein, wash the

resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable

binding buffer. If you need to recharge the resin, see page 17.

Purification Procedure—Hybrid Conditions

Introduction

Materials Needed

ProBond

™

Columns

For certain insoluble proteins, use the Hybrid protocol to restore protein

activity following cell lysis and binding under denaturing conditions. Note that

this procedure will not work for all proteins and should be tested using your

particular recombinant proteins.

Be sure to check the pH of your buffers before starting. Note that the

denaturing buffers containing urea will become more basic over time.

You will need the following items:

• Denaturing Binding Buffer (supplied with the system or see page 19 for

recipe)

• Denaturing Wash Buffer, pH 6.0 (supplied with the system or see page 19

for recipe)

• Native Wash Buffer (page 8 for recipe)

• Native Elution Buffer (page 8 for recipe)

• Prepared ProBond

™

Columns under denaturing conditions (page 11)

• Lysate prepared under denaturing conditions (page 2)

Prepare the ProBond

™

columns using Denaturing Binding Buffer as described

on page 11.

Continued on next page

Purification Procedure—Hybrid Conditions,

Continued

Purification Under

Hybrid Conditions

Using the denaturing buffers, columns and cell lysate prepared under

denaturing conditions, follow the purification procedure below:

1. Add 8 ml of lysate (page 2) to a prepared ProBond

™

Column (page 11).

2. Bind for 15–30 minutes at room temperature using gentle agitation (e.g.,

on a rotating wheel) to keep the resin suspended in the lysate solution.

Settle the resin by gravity or low speed centrifugation (800 × g) and

carefully aspirate the supernatant.

3. Wash the column with 4 ml Denaturing Binding Buffer supplied with the

kit by resuspending the resin and rocking for two minutes. Settle the

resin by gravity or low speed centrifugation (800 × g) and carefully

aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE

analysis. Repeat this step one more time.

4. Wash the column with 4 ml Denaturing Wash Buffer, pH 6.0 supplied

with the kit by resuspending the resin and rocking for two minutes.

Settle the resin by gravity or low speed centrifugation (800 × g) and

carefully aspirate the supernatant. Save supernatant at 4°C for

SDS-PAGE analysis. Repeat this step one more time.

5. Wash the column with 8 ml Native Wash Buffer (page 8 for recipe) by

resuspending the resin and rocking for two minutes. Settle the resin by

gravity or low speed centrifugation (800 × g) and carefully aspirate the

supernatant. Save supernatant at 4°C for SDS-PAGE analysis. Repeat this

step three more times for a total of four native washes.

6. Clamp the column in a vertical position and snap off the cap on the

lower end. Elute the protein with 8–12 ml Native Elution Buffer (see

page 8 for recipe). Collect 1 ml fractions and analyze with SDS-PAGE.

If you wish to reuse the resin to purify the same recombinant protein, wash the

resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable

binding buffer. If you need to recharge the resin, see page 17.

Troubleshooting

Introduction

Review the information below to troubleshoot your experiments with the

ProBond

™

Purification System.

For troubleshooting problems with antibody detection, see the antibody manual

supplied with the system.

Problem Probable Cause Possible Solution

• No recombinant

protein recovered

following elution.

• Nothing bound because • Try denaturing conditions.

of protein “folding.”

• Expression levels too

low.

• Protein washed out by

too stringent washing.

• Not enough sample

loaded.

• Recombinant protein

has very high affinity

for ProBond

™

resin.

• Optimize expression levels using the guidelines

in your expression manual.

• Raise pH of wash buffer in high-stringency wash

step. Wash less extensively in high-stringency

wash step.

• Increase amount of sample loaded or lysate

used.

• Increase stringency of elution by decreasing the

pH or increasing the imidazole concentration.

• To preserve activity, use EDTA or EGTA

(10-100 mM ) to strip resin of nickel ions and

elute the protein.

• Perform all purification steps at 4°C.

• Check to make sure that the 6xHis-tag is not

cleaved during processing or purification.

• Include protease inhibitors during cell lysis.

• Good recombinant-• Wash conditions not

protein recovery but

stringent enough.

contaminated with

• Other His-rich proteins

non-recombinant

in sample.

proteins.

• Lower pH of wash buffer in high-stringency

wash step. Wash more extensively.

• Consider an additional high stringency wash at

a lower pH (i.e., between pH 6 and pH 4) before

the elution step.

• Further purify the eluate on a new ProBond

™

column after performing dialysis of the eluate

against the binding buffer and equilibrating the

column with binding buffer.

• Perform second purification over another type of

column.

• Recombinant protein

• Try denaturing conditions.

has low affinity for

• Try an imidazole step gradient elution.

resin; comes off in wash

• Try a pH gradient with decreasing pH.

with many

contaminating proteins.

Continued on next page

• Protein degraded.

Troubleshooting,

Continued

Problem Probable Cause Possible Solution

• Low recombinant • Recombinant protein not

protein recovery and

binding tightly to resin.

contaminated with

non-recombinant

proteins.

• Try denaturing conditions.

• Try “reverse-chromatography”: bind lysate,

including recombinant protein; allow

recombinant protein to come off in low

stringency washes; collect these fractions; re-

do chromatography on saved fractions on

new or stripped and recharged column.

Works for native purification only.

• Consider an additional high stringency

wash at a lower pH (i.e., between pH 6 and

pH 4) before elution step.

• Load less protein on the column or use more

resin for purification.

• Expression levels too low.

• Some recombinant

protein in the flow

through and wash

fractions

• Column turns

reddish brown.

• Protein overload.

• DTT is present in buffers.

• Use β-mercaptoethanol as a reducing agent.

• Recharge the column as described on

page 17.

• Perform purification at room temperature.

• Add solubilization reagents such as 0.1%

Triton X-100 or Tween-20 or stabilizers such

as Mg

. These may be necessary in all

buffers to maintain protein solubility.

• Run column in drip mode to prevent protein

from dropping out of solution.

• Column turns white. • Chelating agents present in

buffer that strip the nickel

ions from the column.

• Protein precipitates

during binding.

• Temperature is too low.

• Protein forms aggregates.

Appendix

Additional Protocols

Cleavage of the

Fusion Peptide

Recharging

ProBond

™

Resin

If your recombinant fusion protein contains the recognition sequence for

enterokinase (EnterokinaseMax

™

) or AcTEV

™

Protease between the 6xHis-tag

and the protein, you may cleave the 6xHis-tag from the fusion protein using the

specific protease. You can cleave the tag after obtaining the purified

recombinant fusion protein or while the protein is bound to the nickel-chelating

resin.

EnterokinaseMax

™

is a recombinant preparation of the catalytic subunit of

enterokinase. This enzyme recognizes -Asp-Asp-Asp-Asp-Lys- and cleaves

after the lysine. It has high specific activity, leading to more efficient cleavage,

and requires less enzyme.

Description Catalog no.

EnterokinaseMax

™

, 250 units E180-01

EnterokinaseMax

™

, 1000 units E180-02

AcTEV

™

Protease is an enhanced form of Tobacco Etch Virus (TEV) protease

that is highly site-specific, active, and more stable than native TEV protease.

AcTEV

™

Protease recognizes the seven-amino-acid sequence Glu-Asn-Leu-Tyr-

Phe-Gln-Gly and cleaves between Gln and Gly with high specificity.

Description Catalog no.

AcTEV Protease

™

, 1,000 units 12575-015

AcTEV Protease

™

, 10,000 units 12575-023

ProBond

™

resin can be used for up to three or four purifications of the same

protein without recharging. Wash the resin with 0.5 M NaOH for 30 minutes

and equilibrate the resin with the appropriate binding buffer, if you are reusing

the resin.

We recommend not recharging the resin more than three times and only

reusing it for purification of the same recombinant protein. If the resin turns

white due to the loss of nickel ions from the column, recharge the resin.

To recharge 2 ml of resin in a purification column:

1. Wash the column two times with 8 ml 50 mM EDTA to strip away the

chelated nickel ions.

2. Wash the column two times with 8 ml 0.5 M NaOH.

3. Wash the column two times with 8 ml of sterile, distilled water.

4. Recharge the column with two washes of 8 ml NiCl

hexahydrate at a

concentration of 5 mg/ml prepared in sterile, distilled water.

5. Wash the column two times with 8 ml distilled water.

6. Add 0.02% azide or 20% ethanol as a preservative and cap or apply a

parafilm to the column. Store at room temperature.

Recipes

Buffer Stock

Solutions (10X)

5X Native

Purification Buffer

3 M Imidazole

pH 6.0

To prepare the buffer solutions described below, you need to prepare sodium

phosphate stock solutions:

Stock Solution A (10X)

200 mM sodium phosphate, monobasic (NaH

)

5 M NaCl

Dissolve 27.6 g of monobasic sodium phosphate (NaH

) and 292.9 g of NaCl

in 800 ml deionized water. Mix well and adjust the volume to 1 L with

deionized water. Store solution at room temperature.

Stock Solution B (10X)

200 mM sodium phosphate, dibasic (Na

HPO

)

5 M NaCl

Dissolve 28.4 g dibasic sodium phosphate (Na

HPO

) and 292.9 g of NaCl in

800 ml of deionized water. Mix well and adjust the volume to 1 L with

deionized water. Store solution at room temperature.

250 mM NaH

, pH 8.0

2.5 M NaCl

Prepare 200 ml solution as follows:

1. To 180 ml deionized water, add

Sodium phosphate, monobasic 7 g

NaCl 29.2 g

2. Mix well and adjust the pH with NaOH to pH 8.0.

3. Bring the final volume to 200 ml with deionized water.

4. Store buffer at room temperature.

3 M Imidazole

500 mM NaCl

20 mM Sodium Phosphate Buffer, pH 6.0

Prepare 100 ml solution as follows:

1. To 90 ml deionized water, add

Imidazole 20.6 g

Stock Solution A (10X) 8.77 ml

Stock Solution B (10X) 1.23 ml

2. Mix well and adjust the pH to 6.0 with HCl or NaOH as necessary.

3. Bring the final volume to 100 ml with deionized water. If the solution

forms a precipitate, heat solution until the precipitate dissolves.

4. Store buffer at room temperature.

Continued on next page

Recipes,

Continued

Guanidinium Lysis

Buffer

Denaturing Binding

Buffer

Denaturing Wash

Buffer

6 M Guanidine Hydrochloride

20 mM Sodium Phosphate, pH 7.8

500 mM NaCl

Prepare 100 ml solution as follows:

1. To 90 ml deionized water, add

Stock Solution A (10X) 0.58 ml

Stock Solution B (10X) 9.42 ml

Guanidine Hydrochloride 57.3 g

2. Stir the solution until completely dissolved. Adjust the pH to 7.8 using 1

N NaOH or 1 N HCl.

3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 µm

filter (autoclaving the solution will alter the pH of the buffer).

4. Store buffer at room temperature.

8 M Urea

20 mM Sodium Phosphate pH 7.8

500 mM NaCl

Prepare 100 ml solution as follows:

1. To 90 ml deionized water, add

Stock Solution A (10X) 0.58 ml

Stock Solution B (10X) 9.42 ml

Urea 48.1g

2. Stir the solution with gentle heating (50-60°C, do not overheat) until

completely dissolved. When cooled to room temperature, adjust the pH

to 7.8 using 1 N NaOH or 1 N HCl.

3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 µm

filter (autoclaving the solution will alter the pH of the buffer).

4. Store buffer at room temperature.

8 M Urea

20 mM Sodium Phosphate, pH 6.0

500 mM NaCl

Prepare 100 ml solution as follows:

1. To 90 ml deionized water, add

Stock Solution A (10X) 7.38 ml

Stock Solution B (10X) 2.62 ml

Urea 48.1g

2. Stir the solution with gentle heating (50-60°C, do not overheat) until

completely dissolved. Adjust the pH to 6.0 using 1 N NaOH or 1 N HCl.

3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 µm

filter (autoclaving the solution will alter the pH of the buffer).

4. Store buffer at room temperature.

Continued on next page

Recipes,

Continued

Denaturing Elution

Buffer

8 M Urea

20 mM Sodium Phosphate, pH 4.0

500 mM NaCl

Prepare 100 ml as follows:

1. To 90 ml deionized water, add

Stock Solution A (10X) 10 ml

Urea 48.1g

2. Stir the solution with gentle heating (50-60°C, do not overheat) until

completely dissolved. Adjust the pH to 4.0 using 1 N NaOH or 1 N HCl.

3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 µm

filter (autoclaving the solution will alter the pH of the buffer).

4. Store buffer at room temperature.

Frequently Asked Questions

For denatured conditions,

why is Guanidinium used for

lysis of cells?

We have found that guanidinium works better for cell lysis than urea;

however, urea works well for the remaining steps.

Can proteins bind to the resin

The optimal binding range is pH 7.2–7.8. However, we have performed

at a pH lower than 7.8?

purifications with columns equilibrated to pH 6.0. Some proteins bind

well under these conditions and will remain bound to the column

following a pH 6.0 wash.

Can glycine be used instead

of sodium phosphate in the

purification system binding

buffers?

No, because glycine is a competitive ligand for nickel.

People have successfully used:

•

Tris-HCl

Tris-Phosphate

Tris-Acetate

Sodium Acetate

Sodium Borate

MES-NaOH

Pipes-HCl

HEPES

Can I use the resin to purify a

protein with fewer than six

histidine residues?

Is there a cell lysis procedure

that will liberate microsome-

bound proteins for

subsequent purification using

ProBond

™

What are recommended

elution conditions for His-

tagged proteins that are

unstable at a pH<7.0?

What is the importance of

NaCl in the binding buffer?

We have not tried to purify proteins with less than six histidines.

However, if several histidines are near each other, you may be able to

attach the protein to the resin well enough for purification.

If solubility is a problem, you can include up to 0.2% Sarkosyl in the 6M

Guanidinium Lysis Buffer—this should solubilize everything and may

still be compatible with purification on the ProBond

™

columns. In

general, anionic detergents are incompatible with nickel chelating

columns, but up to 0.2% Sarkosyl has been used in some cases.

You can elute with a stepped imidazole gradient at a neutral pH of

7.0-7.5). Use 10 mM imidazole, then 50 mM, 75 mM, and so on until the

protein elutes. Note that more contaminating proteins that would have

been washed off at pH 6.0 will remain on the resin at pH 7.0.

ProBond

™

resin has a net positive charge, and 500 mM NaCl is used to

prevent the nonspecific binding of negatively charged proteins.

References

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1994)

Current Protocols in Molecular Biology Vol. 1. John Wiley and Sons, New York.

Blochlinger, K. and Diggelmann, H. (1984) Mol. Cell Biol. 4: 2929.

Frost, E. and Williams, J. (1978) Virology 91: 39.

Goeddel, D.V. ed. (1991) "Expression in Mammalian Cells." Methods in Enzymology Vol. 185. Academic

Press, San Diego, California.

Graham, F.L. and van der Ebb, A.J. (1973) Virology 52: 456.

Laemmli, U.K. (1970) Nature 227: 680-685.

Lopata, M.A., Cleveland, D.W. and Sollner-Webb, B. (1984) Nucleic. Acids Res. 12: 5707.

Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem., 193: 265-275.

Lowy, D.R., Rands, E. and Scolnick, E.M. (1978) J. Virology 26: 291.

Lewis, W.H., et al. (1980) Somat. Cell Genet. 6: 333.

Maniatas, T., Frisch, E.F. and Sambrook, M.D. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring

Harbor Laboratory, Cold Spring Harbor, New York.

Wigler, M. et al., (1977) Cell 11: 223.

Zhou et al., (1990). Biotechniques, 8(2): 172.

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