Bio-Rad D116698 Airflow Monitor Digital Kit No 9743207 and Guardian Jr Kit No 9743208 Installation Procedure

Nuvia™ IMAC Resin

Instruction Manual

Catalog

numbers

7800800
7800801
7800802
120 03 233
120 02782
12004040
12004039

Please read these instructions prior to using
Nuvia IMAC Resins. If you have any questions or
comments regarding these instructions, contact your
Bio-Rad Laboratories representative.

Table of Contents

Section 1 Introduction ....................................1

Section 2 Product Description ..............................2

Section 3 General IMAC Procedures ........................5

Section 4 Lab-Scale Column Packing ........................6

Section 5 Process-Scale Column Packing .....................9

Section 6 Evaluation of Column Packing .....................11

Section 7 Immobilizing Metal Ions ..........................12

Section 8 Sample Preparation, Purification, and Optimization .....13

Section 9 Regeneration, Cleaning, Sanitizing, and Storing ........16

Section 10 Regulatory Support .............................17

Section 11 Troubleshooting Guide ...........................18

Section 12 Ordering Information ............................20

Section 13 References ...................................21

ii | Nuvia IMAC Resin

Section 1 Introduction

Section 1

Introduction

Immobilized metal affinity chromatography (IMAC) is a powerful purification
technique that relies on a biomolecule’s affinity for metals immobilized onto a
chelating surface. The chelating ligand may be charged with transition metals
such as Ni
in histidine/cysteine residues bind with high selectivity to the metal ions and are
then strongly retained on porous chromatographic supports. The strong affinity
of the molecule for metal ions often makes extensive optimization unnecessary.

Nuvia
purifications. It is compatible with high flow rates and offers superior
binding capacity. It can be easily scaled up from lab- to bioprocess-scale
manufacturing. Refer to the Nuvia IMAC product information sheet (bulletin

6859) and Tables 1, 2, and 3 for more product details.

If you have questions or require method development assistance with Nuvia
IMAC Resin, please contact your local Bio-Rad process chromatography
representative or the Bio-Rad technical support group for assistance at
1-800-4-BIORAD (1-800-424-6723).

2+

, Cu2+, Co2+, or Zn2+. Proteins tagged with histidine or naturally rich

™

IMAC Resin is optimized for high productivity in downstream

Nuvia IMAC Resin | 1

Section 2 Product Description

Section 2

Product Description

Nuvia™ IMAC Resin is based on Bio-Rad’s innovative UNOsphere™ Beads,
which are manufactured using proprietary polymerization and derivatization
technologies (U.S. patent 6,423,666), with nitrilotriacetic acid (NTA) as the
functional ligand. The tertiary amine and carboxylic acid side chains of NTA
serve as the chelating groups for divalent metal ions.

Nuvia IMAC Resin is provided as 50% (v/v) slurry in 20% ethanol or 2% benzyl
alcohol. Multiple pack sizes are available for process- and laboratory-scale use,
in both uncharged and Ni
include Bio-Scale

™

Plates for purification condition screening, and bottles for manufacturing-scale
purifications.

The technical specifications of Nuvia IMAC Resin are listed in Table 1; chemical
compatibility and stability are shown in Tables 2 and 3.

Table 1. Characteristics of Nuvia IMAC Resins.

Property Description

Ligand Nitrilotriacetic acid

Particle size 38–53 µm

Total ligand density ≥18 µmol/ml

Dynamic binding capacity* >40 mg/ml at 300 cm/hr

Compression factor 1.2 0–1.25

Recommended linear flow rate 50–300 cm/hr

Pressure vs. flow performance

pH stability 2–14

Shipping solution 20% ethanol or 2% benzyl alcohol

Regeneration 50 mM EDTA, pH 8.0 (stripping)

CIP solution 1 N NaOH

Sanitization 1 N NaOH

Storage conditions 20% ethanol or 2% benzyl alcohol

Shelf life 5 years

2+

charged versions. The multiple user-friendly formats

Mini Cartridges and prepacked Foresight™ Columns and

Under 2 bar at flow rate of 300 cm/hr in deionized water
(20 x 20 cm packed bed, 1.2 compression factor)

1 N NaOH (CIP/SIP)
100 mM Ni

(recharging)

2SO4

* 10% breakthrough capacity determined with 1.2 mg/ml of a 40 kD histidine-tagged protein

in 50 mM sodium phosphate, 5 mM imidazole, and 300 mM NaCl, pH 7.5 using a
1 ml (0.5 x 5 cm) column

2 | Nuvia IMAC Resin

Section 2 Product Description

Table 2. Chemical compatibility.

Reducing agents* Compatibility concentration

DTE 10 mM

DTT 10 m M

ß-Mercaptoethanol 20 mM

TCEP 20 mM

Reduced glutathione 20 mM

Denaturing agents

Guanidine hydrochloride 6 M

Urea 8 M

Detergents

Triton X-100 (nonionic) 5%

Tween 20 (nonionic) 5%

NP-40 (nonionic) 5%

Cholate (anionic) 5%

SDS (anionic) 5%

Sodium lauroyl sarcosinate (anionic) 2%

Cetyltrimethylammonium bromide (CTAB) (cationic) 5%

CHAPS (zwitterionic) 5%

CHAPSO (zwitterionic) 5%

Additives

Glycerol 50%

Na

2SO4

200 mM

NaCl 2 M

EDTA 2 mM**

EGTA 10 mM

Na citrate 100 mM

Imidazole 500 mM

Ethanol 20%

Ca

Mg

2+

2+

10 mM***

100 mM***

Amino acids Not recommended

(NH

SO

4)2

4

2 M

†

Nuvia IMAC Resin | 3

Section 2 Product Description

Table 2. Chemical compatibility, continued.

Buffer substances Compatibility concentration

NaPi, pH 7.5 100 mM

KPi, pH 7.5 100 mM

HEPES, pH 7.5 100 mM

MOPS, pH 7.5 100 mM

Tris HCl, pH 7.5 100 mM

Buffer pH range ~7–8

* For best results, perform a blank run before loading by washing the column with 5 CV of DI water,

5 CV of elution buffer, and then equilibrating the column with ~5–10 CV of binding buffer

** Static binding capacity for a 45 kD protein ≥20 mg per ml of resin

*** HEPES or Tris buffers should be used to prevent precipitation

† Higher concentrations may cause protein precipitation

Table 3. Chemical stability. The treatments have no effect on binding.

Chemical Treatment

500 mM imidazole* 2 hr, RT, re-equilibration

5 mM DTT* 24 hr, RT, re-equilibration

5 mM TCEP* 24 hr, RT, re-equilibration

20 mM b-ME* 24 hr, RT, re-equilibration

6 M GnHCI* 24 hr, RT, re-equilibration

10 mM EDTA** 72 hr, RT, recharge, re-equilibration

100 mM EDTA** 2 hr, RT, recharge, re-equilibration

100 mM HOAc* 72 hr, RT, re-equilibration

10 mM HCI** 1 week, RT, recharge, re-equilibration

100 mM NaOH** 1 week, RT, recharge, re-equilibration

1 M NaOH** 48 hr, RT, recharge, re-equilibration

* No recharging is needed
** Requires recharging

RT, room temperature

4 | Nuvia IMAC Resin

Section 3 General IMAC Procedures

Section 3

General IMAC Procedures

Nuvia™ IMAC Resin is provided fully hydrated as a 50% (v/v) slurry in
20% ethanol or 2% benzyl alcohol. For column packing, replacing the shipping
solution with packing buffer is recommended. Small volumes of Nuvia IMAC
Resin can be easily washed in a Büchner funnel with 4–5 bed volumes of
water or buffer. For large volume preparation, cycle through 3–4 settling and
decanting steps using water or buffer.

Low concentrations (0.001–15 mM) of imidazole are recommended in
equilibration buffer, which will aid in reducing nonspecific binding of weakly
interacting contaminant proteins. Imidazole levels should be optimized for each
protein based on the concentrations required to prevent nonspecific binding
while enabling target protein adsorption.

Protein Binding

Optimal protein binding with Nuvia IMAC Resin is achieved at pH 7.0–8.0.
Proteins containing engineered histidine tags, as well as untagged proteins
rich in histidine and/or cysteine residues, can bind to Nuvia IMAC Resin with
varying affinities.

Recommended equilibration and binding buffer: 20–50 mM sodium or
potassium phosphate, containing up to 1 M NaCl to reduce nonspecific protein
binding

Washing

The optimal pH and/or imidazole concentration to be used in wash buffers is
protein dependent and should be determined experimentally.

Recommended wash buffer: 5–30 mM imidazole, 50 mM sodium phosphate,
and 300 mM NaCl

Elution

Proteins can be eluted with higher concentrations of imidazole in the elution buffer.

■

Recommended elution buffer:

– 30–500 mM imidazole, 50 mM sodium phosphate, and 300 mM NaCl

Nuvia IMAC Resin | 5

Section 4 Lab-Scale Column Packing

■

Histidine-tagged proteins can also be eluted by:

– Introduction of a competitor ligand in a step or gradient elution with

ligands such as histidine, histamine, or glycine

– Reduction of the pH to 4.5–5.3

– Stripping of the immobilized metal with chelating agents such as

EDTA or EGTA

Section 4

Lab-Scale Column Packing

Medium-Pressure Columns

Slurry packing is preferred for small columns. For best results, use 5–50 mm ID
columns and a bed height of 5–30 cm.

Recommended Columns

Bio-Rad’s Bio-Scale™ MT High-Resolution Columns may be used. They are
convenient to use with Bio-Rad’s NGC
medium- or high-pressure system:

™

Chromatography System or any

■

7510081 – Bio-Scale MT2 Column (7 x 52 mm) for bed volumes
up to 2 ml

■

7510083 – Bio-Scale MT5 Column (10 x 64 mm) for bed volumes
up to 5 ml

■

7510085 – Bio-Scale MT10 Column (12 x 88 mm) for bed volumes
up to 10 ml

■

7510087 – Bio-Scale MT20 Column (15 x 113 mm) for bed volumes
up to 20 ml

Materials

■

Empty column (1–5 cm ID x 30 cm) with flow adaptors, inlet and outlet
ports, glass filter, Nuvia

6 | Nuvia IMAC Resin

™

IMAC Resin, packing reservoir, pump

Section 4 Lab-Scale Column Packing

Resin Preparation

Nuvia IMAC Resins are supplied in a 20% ethanol or 2% benzyl alcohol
solution. Small columns can be packed in this solution, although removal
of storage solution is preferred. Before applying sample, ensure that all the
storage solution is removed during the equilibration step. Ni charged Nuvia
IMAC resin is ready to use. The uncharged resin can be charged according to
the protocol mentioned in Section 7.

Method

1. Eliminate air from the column dead spaces. Attach the inlet of a peristaltic or
other pump to the outlet of the column. Fill the column with distilled water to
about 10% of its volume. Flush end pieces with distilled water to ensure that
the bottom of the bed support is fully saturated and free of air bubbles. Allow
a few centimeters of distilled water to remain when closing the outlet valve.

2. Suspend the resin in a beaker by gently swirling or stirring with a glass or
plastic rod.

3. Carefully transfer about a third of the slurry down the side into the column
using a glass or plastic rod to avoid introducing air bubbles.

4. Start the pump at a low flow rate (for example, 0.5 ml/min). The resin
will begin to pack in the column. As the liquid level in the column drops,
continue to transfer the rest of the slurry until the packed bed reaches
1 cm from the top. Stop or slow the pump flow rate as necessary.

5. Gently add more distilled water down the side of the column to make
sure the liquid does not fall below the resin level. Continue adding distilled
water until the bed seems to have stabilized. Then gently fill the column to
the top with distilled water.

6. Stop the pump.

7. Insert the adaptor into the column at an angle to avoid introducing any air
bubbles. Make sure the exit tubing is open so that distilled water can flow
out the top adaptor along with any air.

8. Adjust the adaptor to sit directly on top of the resin bed.

9. Open the column outlet and pump distilled buffer through the column
at a) a packing flow rate of ~400–600 cm/hr for 5–10 min, or b) the
maximum pressure allowed by the column hardware and resin. The
resin bed will compress while packing at high flow rates. Mark the
compression level with a pen.

10. Stop the flow. At this point, the resin bed height may readjust and rise.
If this happens, adjust the flow adaptor to compress the bed another

0.1–0.5 cm past the level marked with the pen.

Nuvia IMAC Resin | 7

Section 4 Lab-Scale Column Packing

11. Reconnect the pump and equilibrate. Pass eluent (distilled water or
equilibration buffer) through the column at the packing flow rate. During
equilibration, the bed may compress even further. When a constant bed
height is reached, mark the compression level at this flow rate. Again,
adjust the adaptor to compress the bed an additional 0.1–0.5 cm past the
level marked.

Note: Chromatographic steps during purification should not be run at greater
than 75% of the packing flow rate.

Sample Preparation–Size Columns

Equipment

■

Sample preparation–size columns (for example, Micro Bio-Spin™ Columns,
catalog #7326207), plasticware, 2 ml capped and 2 ml capless tubes, Nuvia
IMAC Resin, tabletop centrifuge, 1 ml pipet with wide-bore pipet tips

Method

1. Thoroughly suspend Nuvia IMAC Resin.

2. Place the column into an appropriate collection vessel, for example, a 2 ml
capless collection tube.

3. Using a pipet, transfer the appropriate amount of Nuvia IMAC Resin to a
microcentrifuge tube. If using a Micro Bio-Spin Column, transfer 0.2 ml
slurried 50% v/v Nuvia IMAC Resin to the column. This is equivalent to
~100 µl of a packed resin bed.

4. Centrifuge at 1,000 x g for 15 sec to pack resin and remove storage
solution.

5. Wash column with at least 5 column volumes (CV), or ~500 µl, of distilled
water. Centrifuge at 1,000 x g for 15 sec to pack resin.

8 | Nuvia IMAC Resin

Section 5 Process-Scale Column Packing

Section 5

Process-Scale Column Packing

10 x 20 cm Column

The packing method example shown in this section was done on a 10 x 20 cm
BPG column.

1. Remove the air in the bottom frit of the column with water and then close
the bottom outlet.

2. Calculate the amount of resin required to pack the column at the desired
bed height based on the slurry concentration and compression factor.
Pour the resin slurry (concentration 45–65%) into the column.

3. Allow the resin to settle for 30 min to have a liquid gap of 2–3 cm above
the bed.

4. Lower the top adaptor to 1 cm below the liquid surface. Gently shake the
adaptor to remove the air near the seal and underneath the adaptor.

5. Tighten the seal and lower the adaptor slightly to push out the remaining
air in the adaptor through the waste line of the top valve.

6. Connect the pump to the top valve and open the bottom valve.

7. Consolidate the bed by pumping water through the column at 60 cm/h
until a stable bed is formed.

8. Stop the pump and close the bottom outlet.

9. Wait ~10 min to allow the bed to settle completely.

10. Record the settled bed height and calculate the settled bed volume to
verify the calculation of resin volume.

11. Loosen the seal slightly and lower the top adaptor to 1 cm above the
settled bed surface. Tighten the seal well.

12. Calculate and mark the target bed height based on the target
compression factor.

13. Close the top adaptor outlet and open the bottom outlet.

14. Push down the top adaptor slowly (approximately 100 cm/h) to compress
the bed to the target bed height.

15. Condition the column with 2 CV of water at downward flow of 320 cm/h.

Nuvia IMAC Resin | 9

Section 5 Process-Scale Column Packing

20 x 20 cm Column

The packing method example shown in this section was performed in a
20 x 20 InPlace
decantations.

1. Remove the air in the bottom frit of the column with water and then close
the bottom valve.

2. Calculate the amount of resin required to pack the column at the desired
bed height based on the slurry concentration and the compression factor.
Pour the resin slurry (concentration 45–65%) into the column.

3. Allow the resin to settle for 30 min to have a liquid gap of 2–3 cm above
the bed.

4. Lower the top piston to approximately 1 cm below the liquid surface.
Gently shake the piston to remove the air around the seal and underneath
the piston.

5. Inflate the seal to 4 bars and lower the piston slightly to push out the
remaining air inside the piston through the waste line of the top valve.

6. Close the top valve and open the bottom valve.

7. Set the piston speed to 200 cm/h.

8. Set the target bed height.

9. Start the axial compression.

10. Monitor the piston position during compression to make sure it stops at
the target bed height. Reset the bed height as needed.

11. Close the bottom valve when the compression completes.

12. Inflate the seal to 6 bars.

13. Condition the column with 2 CV of water at downward flow of 200 cm/h
and 23 psi.

™

Column, following removal of bulk ethanol by 3–4 successive

10 | Nuvia IMAC Resin

Section 6 Evaluation of Column Packing

Section 6

Evaluation of Column Packing

Poor column packing can lead to compromised product quality and economics.
Therefore the efficiency of packing must be tested after each column packing.
In addition, packing analysis during process development can assist in setting
appropriate acceptance criteria during scale-up.

After column packing is complete, equilibrate the column with up to 5 CV
equilibration buffer. To test the efficiency of the column packing operation, inject
a sample of a low molecular weight, unretained compound (for example, acetone
or 1 M NaCl) to determine the height equivalent to a theoretical plate (HETP). If
acetone is used as the test marker (use an ultraviolet absorbance monitor set at
280 nm), the equilibration buffer must have a salt concentration <100 mM. If
1 M NaCl is the test marker (use a conductivity monitor), then the equilibration
buffer salt concentration should be 100–200 mM. The recommended sample
volume is 1–2% of the total column volume. Column testing should be operated
using the same linear velocity used to load and/or elute the sample. To obtain
comparable HETP values among columns, the same conditions must be
applied. Minimum theoretical plate values should be 1,000–3,000 plates/m for
linear velocities of 50–600 cm/hr.

HETP = L/N
where
L = Bed height (cm)
N = Number of theoretical plates
Calculation for N = 5.54(V
where
V

= Peak elution volume or time

e

W

= Peak width at peak half height in volume or time

½h

V

and W½h should always be in the same units

e

Reduced plate height can also be used to evaluate column packing efficiency.

e/W½h

2

)

The reduced plate height h is calculated as follows:

h = HETP/d where d is the diameter of the beads

Peak asymmetry factor calculation:

A

= b/a

s

a = Front section of peak width at 10% of peak height bisected by line
denoting V

e

b = Back section of peak width at 10% of peak height

Nuvia IMAC Resin | 11

Section 7 Immobilizing Metal Icons

Section 7

Immobilizing Metal Ions

Protein selectivity may be optimized through the choice of metal ion used.
Uncharged Nuvia
targeted to a specific protein. The protocol for immobilizing a metal ion is
shown below; for initial screening, typically Ni

1. After column packing is complete, the column is ready for the removal or
addition of metal ions.

2. If necessary, strip any metal ion by washing with 10 CV of 50 mM sodium
phosphate, 300 mM NaCl, and 50 mM EDTA at pH 8.0. Any color due to
the presence of metal ions should be eliminated by this step.

3. Equilibrate the column with 5 CV of 50 mM sodium acetate at pH 4.5.

4. Prepare a 100 mM solution of the metal ion of choice. For best results, the
pH of the solution should be <7.

5. Apply 3 CV of the metal ion solution.

6. Wash with 5 CV of 50 mM sodium acetate at pH 4.5.

7. Wash with 10 CV of deionized water.

8. Equilibrate with at least 5 CV of starting buffer for the purification.

Note: Uncharged resin will be white; charged areas will be colored.

™

IMAC Resin can be used to select the best metal ion

2+

or Cu2+ is chosen:

12 | Nuvia IMAC Resin

Section 8 Sample Preparation, Purification, and Optimization

Section 8

Sample Preparation, Purification,
and Optimization

Preparation

The sample should be free of particulate matter prior to application. This can
be achieved by centrifugation or filtration. The choice of binding buffer will
vary based on the sample properties. Sodium or potassium phosphate is
recommended as a general starting buffer. The choice of elution buffer will
vary depending on the procedure used. For example, a range of imidazole
concentrations (30–500 mM) may be used to elute bound protein from

™

Nuvia

IMAC Resin. As an alternative to imidazole elution, the pH can be
lowered to within the range of 2.5–7.5. (Note: below pH 4, metal ions will be
stripped off the medium.) Chelating agents such as EGTA or EDTA can also be
used to dissociate the protein from the resin. (Note: with chelating agent, metal
ions will be stripped off with the protein.) Suggested buffers are shown below.

Binding/Wash Buffer: 50 mM sodium phosphate at pH 8.0 with 300 mM NaCl
and 5–30 mM imidazole. If protein binding is weak, reduce the concentration
of imidazole

Elution Buffer: 50 mM sodium phosphate at pH 8.0 with 300 mM NaCl and
30–500 mM imidazole

Purification Protocol for a Packed Column

1. Equilibrate the column with at least 5 CV of binding buffer.

2. Apply the sample onto the column using the desired flow rate.

3. Wash the resin with at least 5 CV of wash buffer to remove unbound

samples until the absorbance at 280 nm is at or near baseline.

4. Collect fractions from the wash step (unbound proteins).

5. Elute bound proteins either using a step change in buffer or as a

linear gradient.

6. Collect fractions from the elution step (bound proteins).

7. The collected fractions can be further analyzed using absorbance at A

SDS-PAGE, ELISA, etc.

Nuvia IMAC Resin | 13

280

,

Section 8 Sample Preparation, Purification, and Optimization

Purification Protocol for a Spin Column

Part 1. Binding the sample

1. Place prepacked spin column in an appropriate spin collection tube.

2. Pre-equilibrate the spin column with 5 CV of binding buffer.

3. Add an appropriate amount of the sample (≤0.5 ml) to the micro spin

column.

4. Mix by pipetting up and down 5 times. Incubate for up to 30 min in micro

spin column.

5. Centrifuge at 1,000 × g for 1 min to remove the unbound proteins.

Part 2. Washing the sample

1. Insert the micro spin column into a new, clean collection vessel.

2. Wash the resin with at least 5 CV of wash buffer. Pipet up and down at

least 5 times.

3. Centrifuge at 1,000 × g for 1 min to remove remaining unbound proteins.

The wash step can be repeated if necessary.

Part 3. Eluting the sample

1. Insert the micro spin column into a new, clean collection vessel.

2. Elute bound proteins with 5 CV of elution buffer. Pipet up and down at

least 5 times and incubate for up to 5 min.

3. Centrifuge at 1,000 × g for 1 min to remove bound proteins.

4. Analyze fractions from above steps using absorbance at A

ELISA, etc.

, SDS-PAGE,

280

14 | Nuvia IMAC Resin

Section 8 Sample Preparation, Purification, and Optimization

Optimization

For optimal protein purification, it is crucial that the imidazole concentration in
the sample and the binding, elution, and wash buffers be empirically established.
Gradient elution tests using a gradient mixer coupled to a chromatography system,
such as the NGC

■

For the binding buffer, the concentration of imidazole can be started at
20 mM. If large amounts of contaminants are also adsorbed onto the
resin, the concentration of imidazole in the sample and equilibration buffer
may be increased. This may reduce the overall amount of target protein
bound. However it will also increase the column’s binding capacity for the
target protein due to the reduction in contaminating proteins

■

For the wash step, use an imidazole concentration slightly lower than
the concentration necessary to elute the target protein. This will increase
purity by removing nonspecifically bound contaminants without eluting the
target proteins

■

The elution buffer should contain imidazole corresponding to the
concentration required to elute the target protein

™

System, can be used to optimize imidazole concentrations.

Nuvia IMAC Resin | 15

Section 9 Regenerating, Cleaning, Sanitizing, and Storing

Section 9

Regenerating, Cleaning, Sanitizing,
and Storing

Nuvia™ IMAC Resin should be cleaned prior to reuse. The extent of cleaning
depends on the downstream application.

Regenerating the Medium

Regenerate metal-charged Nuvia IMAC Resins by first stripping with an EDTA
solution. Wash the column with 10 CV of 50 mM sodium phosphate, 300 mM
NaCl, and 50 mM EDTA at pH 8.0. Ensure that residual EDTA is completely
removed from the column by washing it with 3–5 CV of binding buffer followed
by 3–5 CV of distilled water. Equilibrate the column with 5 CV of 50 mM
sodium acetate at pH 4.5. Recharge with the metal ions as described in
Section 7.

Cleaning in Place

Wash the column with the following solution.

■

1 N NaOH up to 3 hr (removes precipitated, hydrophobic, and
lipoproteins)

– Exposure time is usually 1–3 hr

– Rinse with 10 CV of distilled water

Alternatively, other cleaning-in-place solutions can be used.

■

2 M NaCl (removes ionic contaminants)

– 10–15 min exposure time

– Rinse with 10 CV of distilled water

■

70% ethanol or 30% isopropyl alcohol (removes precipitated,
hydrophobic, and lipoproteins)

– 15–20 min exposure time

– Alternatively, use 0–30% gradient isopropyl alcohol over 5 CV,

followed by 2 CV of 30% isopropyl alcohol

– Rinse with 10 CV of distilled water

16 | Nuvia IMAC Resin

Section 10 Regulatory Support

Remove cleaning solution(s) from column by rinsing with 10 CV of binding buffer
(for example, 50 mM sodium phosphate, 300 mM NaCl, pH 8).

■

Column cleaning can be monitored by UV signal. Ensure the eluate is at
pH ~8 and the UV signal has returned to baseline

Sanitization

The column may be sanitized with 1 N NaOH.

Rinse solution from column with 3–5 CV of distilled water. Re-equilibrate the
column with 3–5 CV of binding buffer.

Storage

Nuvia IMAC Resin is stable at room temperature across a broad pH range
(2–14). The media may also be stored in either of the following solutions:

■

2% benzyl alcohol

■

20% ethanol

Uncharged resin can be stored with 0.1 NaOH.

Section 10

Regulatory Support

A regulatory support file is available for Nuvia™ IMAC Resin. If you need
assistance validating the use of Nuvia IMAC Resin in a production process,
contact your local Bio-Rad representative.

Nuvia IMAC Resin | 17

Section 11 Troubleshooting Guide

Section 11

Troubleshooting Guide

Possible Cause Solution

Sample is too viscous

High concentration of host nucleic
acids in lysate

Insufficient amount of
homogenization buffer

Sample application causes column to clog

Insufficient clarification of sample Prevent cell debris from clogging the column by increasing

Clogs can also result from protein
precipitation.

No protein is eluted

Expression of target protein in
extract is very low and is not found
in the eluate

Target protein is found in inclusion
bodies or possible insufficient lysis

Target protein is found in the
flowthrough

Elution conditions are too mild or
protein may be in an aggregated
or multimer form

Viscosity of extract can be reduced by nuclease treatment

Dilute sample by adding more homogenization buffer

the centrifugation speed and/or filtering the sample

Consider the use of additives to improve protein stability and
solubility

Check expression level of protein by estimating the amount
in the extract, flowthrough, eluted fraction, and pellet upon
centrifugation. Use western blotting (with anti-6x histidine
antibodies and/or target protein–specific antibodies) ELISA,
or enzyme activity determination

Apply larger sample volume

Minimize contact with hydrophobic surfaces (such as
polyst yrene tubes). Proteins at low concentration may bind
to the surface of the tube

Increase intensity/duration of disruption and homogenization

If protein is insoluble, use 6 M guanidine HCl or 8 M urea to
lyse denatured proteins

Reduce imidazole concentration in sample and binding/wash
buffers. An imidazole gradient may be used to determine
optimal concentrations for wash and elution conditions

Check pH levels of sample. A decrease in pH may result
during the homogenization step or during growth of the
culture medium. Adjust pH to 7–8

The histidine tag may not be accessible

Use denaturing conditions to purify protein or reclone the
plasmid construct with the histidine-tagged sequence
placed at the opposite terminus

Proteoly tic cleavage during fermentation or purification
has caused the histidine tag to be removed. Add protease
inhibitors or make a new construct with histidine tag
attached to other terminus

Increase the concentration of imidazole in elution buffer;
optimize elution buf fer pH

18 | Nuvia IMAC Resin

Section 11 Troubleshooting Guide

Possible Cause Solution

Protein precipitates during purification

Temperature is too low Perform the purification at room temperature

Aggregate forms Add solubilization agents to samples and/or buffers: 0.1%

Triton X-100, Tween 20, 20 mM b-mercaptoethanol, and
≤20% glycerol to improve protein solubility

Poor recovery of target protein

Protein is found in the flowthrough See recommendations in No protein is eluted section, above

Binding capacity of the
column has been exceeded

Target protein was detected in the

Increase the column size or reduce the sample volume
application

Capillary sample loop is too small

flowthrough

Strong adsorption of the target
protein to the matrix

Reduce hydrophobic adsorption by including detergents
or organic solvents, or by increasing the concentration of
imidazole and/or NaCl

Histidine-tagged protein is not pure

Contaminants elute with
target protein

Make binding and wash steps more stringent. Include
10–20 mM imidazole in binding and wash buffers

Prolong the imidazole-containing wash step

™

Column is too large; reduce amount of Nuvia

IMAC Resin

used

Strongly bound contaminants
elute with protein

Very high concentrations of imidazole will cause strongly
bound contaminants to elute as well. Reduce the imidazole
concentration during the elution

Association of contaminating
proteins with target protein via
disulfide bonds

Association between the
histidine-tagged protein and
protein contaminant

Include ≤20 mM b-mercaptoethanol

Note: Exercise caution if using DTT

Add nonionic detergent or alcohol (that is, Triton X-100,
Tween 20, or glycerol) to reduce hydrophobic interactions.
Concentration of NaCl may be increased to minimize
electrostatic interactions

Potential degradation of fusion
protein by proteases

Contaminants exhibit similar
affinity to target protein

Include protease inhibitors in lysis buffer to reduce partial
degradation. Consider performing purification at 4ºC

Add additional chromatography steps; that is, ion exchange,
hydrophobic interaction, or size exclusion

Nuvia IMAC Resin | 19

Section 12 Ordering Information

Section 12

Ordering Information

Catalog # Description*

Bottles

7800800 Nuvia IMAC Resin, 25 ml bottle, Ni-charged
7800801 Nuvia IMAC Resin, 100 ml bottle, Ni-charged
7800802 Nuvia IMAC Resin, 500 ml bottle, Ni-charged
12003233 Nuvia IMAC Resin, 5 L, Ni-charged
120 02782 Nuvia IMAC Resin, 10 L, Ni-charged
12004040 Nuvia IMAC Resin, 5 L
12004039 Nuvia IMAC Resin, 10 L

Prepackaged Formats

120 04 051* * Foresight Nuvia IMAC RoboColumn Unit, 200 µl, Ni-charged
120 04 052** Foresight Nuvia IMAC RoboColumn Unit, 600 µl, Ni-charged
12004035*** Foresight Nuvia IMAC Plates, 20 µl, Ni-charged
12004038 Foresight Nuvia IMAC Column, 1 ml, Ni-charged
12004037 Foresight Nuvia IMAC Column, 5 ml, Ni-charged
7800811 Bio-Scale Mini Nuvia IMAC Cartridge, 1 x 5 ml column, Ni-charged
7800812 Bio-Scale Mini Nuvia IMAC Cartridges, 5 x 5 ml columns, Ni-charged

* Larger quantities available upon request
** Package size: one row of eight columns
*** Package size: 2 x 96-well plates

20 | Nuvia IMAC Resin

Section 13 References

Section 13

References

Bio-Rad (2016). Optimized resin for high productivity in downstream purification processes.
Product information sheet. Bio-Rad Bulletin 6859.

Block H et al. (2009). Immobilized-metal affinity chromatography (IMAC): A review. Methods Enzymol.
463, 439–473.

Hochuli E (1988). Large-scale chromatography of recombinant proteins. J Chromatogr 444, 293–302.

Porath J et al. (1975). Metal chelate affinity chromatography, a new approach to protein fractionation.
Nature 258, 598–599.

Nuvia IMAC Resin | 21

RoboColumn is a trademark of Atoll GmbH. Triton is a trademark of
Dow Chemical Company. Tween is a trademark of ICI Americas, Inc.

Bio-Rad
Laboratories, Inc.

Life Science
Group

D116698 Ver B US/EG

Web site bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800
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16-1212 0417 Sig 1216