Bio-Rad IMAC Resin User Manual
Bio-Rad® Nuvia™ IMAC
Resin
Instruction Manual
Catalog numbers
780-0800
780-0801
780-0802
Please read these instructions prior to using Bio-Rad 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
Nuvia™ IMAC Resins and
UNOsphere™ Technology .......................................... 2
Chemical Interactions ............................................... 3
Resin Characteristics ................................................ 4
Chemical Compatibilities ........................................... 5
Section 3 General IMAC Procedures ..................................... 7
Protein Binding ......................................................... 7
Washes .................................................................... 8
Elution ...................................................................... 8
Purification under Denaturing Conditions .................. 9
Imidazole Concentrations ........................................10
Section 4 Column Packing — Medium-Pressure
Columns .................................................................11
Slurry Packing an IMAC Column...............................11
Recommended Columns..........................................11
Materials ..................................................................11
Resin Preparation ....................................................12
Method ................................................................... 12
Section 5 Column Packing — Sample Preparation–Sized
Columns ................................................................ 14
Materials ................................................................. 14
Resin Preparation ................................................... 14
Method ...................................................................15
Section 6 Immobilizing Metal Ions ........................................16
Section 7 Medium-Pressure Column Purification of
Histidine-Tagged Proteins....................................17
Materials .................................................................17
Method ...................................................................18
Materials and Method for On-Column
Renaturation ........................................................... 20
Section 8 Medium-Pressure Column Purification — Using
an Imidazole Gradient to Determine Optimal
Purification of Histidine-Tagged Proteins ........... 21
Materials ................................................................. 21
Method ................................................................... 22
Section 9 Sample Preparation–Sized Spin-Column
Purification of Histidine-Tagged Proteins ........... 25
Materials .................................................................25
Method ...................................................................26
Section 10 Regenerating, Cleaning, Sanitizing,
and Storage ..........................................................28
Regenerating the Medium .......................................28
Cleaning in Place ...................................................28
Sanitization..............................................................30
Storage .................................................................. 30
Section 11 Troubleshooting Guide ......................................... 31
Section 12 Ordering Information ........................................... 34
Section 13 References ........................................................... 34
Section 14 Legal Notices ...................................................... 34
Section 1
Introduction
Immobilized metal affinity chromatography (IMAC) is a powerful
purification technique that relies on a molecule’s affinity for certain
metals immobilized onto a chelating surface. The chelating
ligand, nitrilotriacetic acid (NTA) in this case, may be charged with
transition metals such as Cu2+, Ni2+, Co2+, or Zn2+. This results in
high selectivity for proteins with clustered histidine residues to be
strongly retained on a porous chromatographic support.
The use of IMAC to separate an expressed recombinant protein
fused with a hexahistidine peptide tag was demonstrated
by Hochuli (1988) to yield a highly purified protein in a single
chromatographic step under both denaturing and native
conditions. The strong affinity of a histidine-tagged molecule for
metal ions often makes extensive optimization unnecessary while
also allowing chromatography under conditions that denature
proteins. For this reason, expression and IMAC purification of
histidine-tagged proteins is frequently used for structural and
functional studies of proteins.
Nuvia IMAC Ni-Charged Resin 1
Section 2
Product Information
Nuvia™ IMAC Resins and UNOsphere™ Technology
Nuvia IMAC resin, a unique affinity support, is based on Bio-Rad’s
innovative UNOsphere beads, which use proprietary polymerization
and derivatization technologies.* The UNOsphere technology
enables the polymeric high-capacity IMAC resin to exhibit excellent
flow properties without compromising protein binding, recovery, or
purity.
Nuvia IMAC uses NTA as its functional ligand. The tertiary amine
and carboxylic acid side chains of NTA serve as the chelating
ligands for divalent metal ions. The structure offers selective
binding of recombinant histidine-tagged proteins when this resin is
charged with Ni2+ or other transition metals. As a result, the desired
proteins can often be purified close to homogeneity in a single
step.
Structural characteristics such as the polymeric nature, optimized
ligand density, and open-pore structure of Nuvia IMAC beads
result in superb mechanical strength with high stringency, low
nonspecific effects, and the ability to provide separations at fast
flow rates. These unique features of the UNOsphere base matrix
lend a number of performance benefits to the Nuvia IMAC resin.
Nuvia IMAC is also stable across a wide pH range (2–14) and
is compatible with most reagents commonly used in protein
purifications, such as denaturants, detergents, and reducing
agents. It is amenable to separations under native or denaturing
conditions using liquid chromatography instrumentation, gravity
flow columns, or sample-preparation spin columns.
Note: UNOsphere media, from which Nuvia IMAC is derived,
was designed to achieve the highest productivity (grams of
drug or target per operational hour per liter of support) possible.
UNOsphere media may be run at the highest rates and loading
capacities and will stay within the pressure limits of the column and
chromatography system.
* U.S. patent 6,423,666.
2 Nuvia IMAC Ni-Charged Resin
Under optimized conditions, the binding capacity for 6x histidinetagged proteins is >40 mg/ml resin (see Table 1). The product is
a 50% (v/v) slurry of resin, which is suspended in a 20% ethanol
solution. Nuvia IMAC resin is amenable to process and laboratoryscale use and is available precharged with Ni2+ in bottles as well
as prepacked into columns. Table 1 lists key characteristics of the
resin, while Table 2 lists a variety of compounds compatible with
Nuvia IMAC support.
Chemical Interactions
Nuvia IMAC resin is composed of NTA groups coupled to
a UNOsphere base matrix via a proprietary polymerization
derivatization technology. It is well-suited to recombinant histidinetagged purifications and results in high binding capacity and
specificity for the target molecule.
Although the most commonly used metal ion for histidine-tagged
purifications is Ni2+, other metals may be used to increase efficacy
of purification. Therefore, choosing another type of immobilized
metal ion can change the selectivity of an IMAC resin.
Fig. 1. Partial structure of Nuvia Ni-charged IMAC resin. Image illustrates UNOsphere base
bead with coupled NTA functional ligand.
Nuvia IMAC Ni-Charged Resin 3
Resin Characteristics
The characteristics of Nuvia IMAC resin are detailed in Table 1.
Table 1. Characteristics of Bio-Rad Nuvia IMAC Resin.
Functional ligand Nitrilotriacetic acid (NTA)
Base bead UNOsphere base matrix
Form 50% suspension in 20% ethanol or pre-
packed into columns; comes precharged
2+
with Ni
Particle size 38–53 µm
Mean particle size 50 µm
Metal ion capacity ≥18 µmol Cu2+/ml Nuvia IMAC resin
Dynamic binding
capacity*
Recommended linear
flow rate
Maximum operating
pressure
pH stability, short-term/
cleaning
Chemical compatibility See Table 2
Storage 4°C to ambient temperature
Shelf life in 20% ethanol ≥3 year at ambient temperature
Operational temperature 4–40°C
Autoclaving conditions 0.1 M sodium acetate at 120°C for 30 min
≥40 mg/ml resin
<500 cm/hr at 25°C
45 psi
2–14
* Dynamic binding capacity conditions (Q10% determination):
Column volume: 1 ml, 5.6 mm x 4 cm (ID x H)
Sample: 1.0 mg/ml 6x histidine-tagged pure protein (40 kD)
Note: Dynamic binding capacity will vary from protein to protein.
4 Nuvia IMAC Ni-Charged Resin
Chemical Compatibilities
The chemical characteristics of Nuvia IMAC resin are detailed in Table 2.
Table 2. Chemical Compatibilities for Nuvia IMAC Resins.
Reagent
Group
Buffer
reagents
Chelating
reagents
Sulfhydryl
reagents
Reagent Comments Stability
Tris, HEPES,
MOPS
Sodium or
potassium
phosphate
EDTA, EGTA Strips nickel ions
β-Mercaptoethanol
Used with
proteins more
stable in
nonphosphate
buffers
from the resin
Reduces random
disulfide bonds
preventing
protein
aggregation
during purification
≤50 mM
secondary and
tertiary amines
50 mM sodium
or potassium
phosphate are
recommended
as starting
buffers
≤0.1 mM
successfully
used to remove
trace metal
contaminants
>1 mM can
cause significant
reduction in
binding capacity
≤20 mM
DTT, TCEP
Detergents Nonionic
detergents (Triton,
Tween)
Zwitterionic
detergents
(CHAPS, CHAPSO)
Denaturants Guanidine HCl
(GuHCl)
Urea
Transition metals
at the center of
IMAC resin (Ni2+)
are susceptible to
reduction
Removes
background
proteins
and nucleic acids
Solubilizes
membrane
proteins
Solubilizes
proteins
Nuvia IMAC Ni-Charged Resin 5
≤10 mM DTT
and 20 mM
TCEP
≤2%
≤1%
≤6 M
≤8 M
Table 2. Chemical Compatibilities for Nuvia IMAC Resins, Continued.
Reagent
Reagent Comments Stability
Group
Other
additives
NaCl Deters
nonspecific
protein binding
due to ionic
interactions
MgCl
2
Essential
component for
purification of
Ca2+ binding
proteins
CaCl
2
Essential
metal cofactor for
nucleases
≤2 M (at least
300 mM NaCl
should be
included in
buffers)
≤100 mM
(HEPES or Tris
buffers should be
used to prevent
precipitation)
≤5 mM (HEPES
or Tris buffers
should be used
to prevent
precipitation)
Glycerol Included
to prevent
hydrophobic
interactions
between proteins
≤20%
(backpressure
may increase
significantly,
slower flow rates
may be required)
Ethanol Included
≤20%
to prevent
hydrophobic
interactions
between proteins
Imidazole Competes for
binding sites with
histidine-tagged
residues by
interaction
with the metal
residues
May be
used in low
concentrations
in the wash
buffer (<30 mM)
to limit binding
of undesired
proteins; for
elution, ≤500 mM
may be used
6 Nuvia IMAC Ni-Charged Resin
Section 3
General IMAC Procedures
Protein Binding
Protein adsorption to immobilized ions is performed around
neutral to slightly alkaline pH conditions (pH 7.0–8.0). To reduce
nonspecific ionic effects, concentrations of up to 1 M NaCl may
be added to the binding solution. Recombinant 6x histidine tags,
located at either the amino or carboxyl terminus of the protein, can
bind with high affinity to the matrix even when the 6x histidine tag
isn’t completely accessible. In general, the fewer the number of
accessible histidine residues, the weaker the protein binding is to
the affinity matrix. Untagged proteins that have naturally occurring
and noncontiguous histidine residues also bind to IMAC resins, but
with much lower affinity.
Batch mode binding is a good alternative if proteins are expressed
at low levels or if the overall concentration of the recombinant
6x histidine tag is low. In this case, proteins are bound to the
Nuvia™ IMAC resin in solution prior to packing the protein-resin
complex into a liquid chromatography column for wash and elution
steps. Altering the imidazole concentration of the lysis buffer may
also optimize binding. Low concentrations (0–15 mM imidazole)
are recommended and will aid in reducing nonspecific binding of
weakly interacting proteins.
Many additives can be used without affecting the binding of
histidine-tagged proteins to IMAC resins. For example, urea,
GuHCl, nonionic detergents, and organic solvents (refer to
Section 2, Table 2) are all valid options. Chelating agents, such as
EDTA or citrate, should not be included. Reducing agents such as
β-mercaptoethanol and DTT may be used at low concentrations.
Potassium phosphate or sodium phosphate buffers are
recommended solutions for equilibration and binding.
Recommended binding buffer:
• 20–50 mM sodium or potassium phosphate, containing up to
1.0 M NaCl.
Begin with: 50 mM sodium phosphate, 0.3 M NaCl, pH 8.0
Nuvia IMAC Ni-Charged Resin 7
Washes
Stringency of 6x histidine-tag binding can be effectively increased
by 1) including low concentrations of imidazole in the binding and
wash solutions, or 2) reducing the pH. Generally, highly expressed
proteins, such as those from a bacterial expression system, have
fewer contaminant proteins that copurify along with the protein of
interest. Endogenous protein contaminants are more abundant
in eukaryotic expression systems and tend to bind to the IMAC
adsorbent more weakly. In these instances, nonspecific binding
of proteins containing neighboring histidine residues becomes
a problem. These endogenous species may be washed from
the resin by either lowering the pH to 6.3 or adding imidazole to
binding and wash solutions in concentrations of 5–30 mM. The
optimal pH and/or imidazole concentration used in wash buffers
is always protein dependent and should always be determined
experimentally.
Recommended wash buffer:
• 5–30 mM imidazole; for example, 50 mM sodium phosphate,
0.3 M NaCl.
Begin with: 5 mM imidazole, 50 mM sodium phosphate,
0.3 M NaCl, pH 8.0
Elution
Desorption of the histidine-tagged protein may be accomplished in
one of three ways: introduction of a competitor ligand, reduction of
the pH, or stripping of the immobilized metal.
In competitive elution, a step or gradient elution with ligands such
as imidazole, histidine, histamine, or glycine may be carried out.
When using a gradient elution with imidazole, it is important to preequilibrate the column with low concentrations of imidazole (1 mM)
and include the same concentration in the sample. This prevents
adsorption of imidazole onto the resin from triggering a drop in pH,
which might prematurely elute bound histidine-tagged proteins.
Lowering the pH of the elution buffer (pH 4.5–5.3) also releases
bound histidine-tagged proteins. In this case, the histidine residues
become protonated and are unable to bind to the immobilized ion.
Protein sensitivity to low pH ranges, however, must be taken into
consideration.
8 Nuvia IMAC Ni-Charged Resin
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