Bio-Scale™Mini
Profinity™IMAC
Cartridges, 1 and 5 ml
Instruction Manual
Catalog #
732-4610
732-4612
732-4614
Table of Contents
Section 1...Introduction........................................1
Section 2 Product Information............................2
Section 3 Connection to Low-Pressure
Chromatography Systems..................6
Section 4 Connection to Medium and High-
Pressure Chromatography Systems...8
Section 5...Buffers and Methods ..........................9
Section 6 Quick Solubility Screening
Protocols..........................................11
Section 7 Preparation of E. coli Lysates ...........15
Section 8 Preparing a Cartridge, and
Subsequent Purification....................17
Section 9 Scaling Up........................................21
Section 10 Regenerating, Cleaning,
Sanitizing, and Storage.....................22
Section 11 Troubleshooting Guide......................25
Section 12 Ordering Information.........................27
Section 13 References.......................................28
Section 14 Legal Notices ...................................29
Section 1
Introduction
Bio-Scale Mini IMAC cartridges are convenient,
disposable, prepacked low-pressure chromatographic
cartridges. Bio-Scale Mini cartridges offer both
increased run-to-run uniformity and high purity of
protein through a patent pending column design
and novel resin technology. Compatible with aqueous
buffers most commonly used for protein purification
the Bio-Scale Mini cartridges offer improved
performance for your protein separation needs.
Immobilized metal affinity chromatography (IMAC) is an
excellent chromatography technique for purification of
His-tagged proteins. The principle of IMAC is based
on the affinity histidine has for metal ions. Side chains
on the iminodiacetic acid (IDA) functional ligand
selectively bind recombinant His-tagged proteins when
the resin is charged with Ni
2+
or other metals. The
advantage of this technique is that proteins can often
be purified close to homogeneity in a single step.
Bio-Scale Mini IMAC cartridges are packed with
1
Bio-Rad’s innovative Profinity™ IMAC resin.
Structural characteristics such as the polymeric
nature, optimized ligand density, and open pore
structure of the Profinity IMAC bead result in superb
mechanical strength with high stringency, low
nonspecific binding, and the ability to perform separations at high flow rates
.
Section 2
Product Information
The Bio-Scale Mini cartridges are disposable,
easy-to-use, prepacked chromatographic cartridges
supplied ready for use in convenient 1 ml and 5 ml
sizes. Cartridges are available for a variety of
chromatographic techniques, including desalting ion
exchange (IEX), affinity (AC), mixed-mode, and
hydrophobic interaction chromatography (HIC). See
Ordering Information, Section 12, for a listing of the
complete Bio-Scale Mini cartridge product line.
The Bio-Scale Mini cartridges are quickly connected
to liquid chromatography systems or luer syringes.
2
The cartridges can be used with any liquid
chromatography system capable of setting a high
pressure limit of 45 psi (equivalent to 3 bar or 300 kPa).
Alternatively, luer fittings offer convenient connection
directly to a Luer-Lok syringe for quick, one-step
purification.
Table 1 Bio-Scale Mini IMAC Cartridge
Specifications
Sizes 1 ml and 5 ml bed volumes
Dimensions 1 ml: 40 mm length x 5.6 mm inner
diameter
5 ml: 40 mm length x 12.6 mm
inner diameter
Maximum pressure tolerance 45 psi
Recommended flow rates 1 ml: 1–2 ml/min (240 cm/hr–480 cm/hr)
5 ml:5–10 ml/min (240 cm/hr–480 cm/hr)
Maximum flow rate 1 ml: 6 ml/min (1440 cm/hr)
5 ml: 20 ml/min (963 cm/hr)
Fittings Female luer-lock inlet and
male luer-lock outlet
Column material Polypropylene
Frit material Polyethylene (HDPE)
Shipping conditions 20% ethanol
Storage recommendations 20% ethanol
Autoclavability Not autoclavable
3
Table 2. Profinity IMAC Resin Specifications
Functional ligand IDA
Base bead UNOsphere
Particle size range 45–90 µm
Mean particle size 60 µm
Metal ion capacity 12–30 µmol Cu
2+
/ml
Dynamic binding capacity* ≥15 mg/ml
Recommended linear flow rate 480 cm/hr
Maximum operating pressure 45 psi
Chemical compatibility See table
Storage 4°C to ambient temperature
Shelf life in 20% EtOH >1 year at ambient temperature
Operational temperature 4–40°C
* Q10% determination of 1.8 mg/ml (His)6-tagged pure protein (32 kD).
Note: Dynamic binding capacity will vary from protein to protein.
Profinity IMAC cartridges are compatible with aqueous
buffers most commonly used with IMAC purification
techniques. For a complete list of chemical
compatibilities, refer to the Profinity IMAC
instruction manual, online at http://www
.biorad.com/cmc_upload/Literature/164111/10001677
B.PDF.
4
Table 3. Buffer and Chemical Compatibilities for
Profinity IMAC cartridges
Reagent Stability
Buffer Reagents
Tris 50 mM
HEPES 50 mM
MOPS 50 mM
Sodium or potassium phosphate 50 mM
Chelating Agents
EDTA, EGTA 0.1 mM
Sulfhydryl Reagents
β−Mercaptoethanol 30 mM
DTT 5 mM
TCEP 10 mM
Detergents
Nonionic detergents (Triton, Tween, NP-40) 5%
Cationic detergents (CTAB) 1% (care must be taken to
avoid protein precipitation)
Zwitterionic detergents (CHAPS, CHAPSO) 5%
Anionic detergents (SDS, Sarkosyl) 1%
Denaturing Agents
Guanidine-HCl 6 M
Urea 8 M
Other Additives
NaCl 2 M (at least 300 mM NaCl
should be included in buffers)
MgCl
2
100 mM (HEPES or Tris
should be used to prevent
precipitation)
CaCl
2
10 mM (HEPES or Tris
should be used to prevent
precipitation)
Glycerol 20%
Ethanol 20%
Imidazole 25 mM in wash buffer
500 mM for elution
Citrate 80 mM
* Profinity IMAC binding capacities are unaffected with typical reagents used for
His-tagged protein purification, up to the concentrations given.
5
Section 3
Connection to
Low-Pressure
Chromatography Systems
Bio-Scale Mini cartridges are ideal for use with any
low-pressure chromatography system, including
Bio-Rad’s BioLogic LP system, Econo gradient
pump, and Model EP-1 Econo pump. For optimum
performance, we recommend choosing biocompatible
low-pressure tubing with an inner diameter (ID) of
1.6 mm.
6
Fig. 1. Luer fittings and column: a cartridge should be
mounted vertically with the arrow on the cartridge pointing
downward.
To order compatible polypropylene 1.6 mm barb to
male and female luer end fittings, refer to the
ordering information located in Section 12 of this
manual.
7
Section 4
Connection to Medium
and High-Pressure
Chromatography Systems
Bio-Scale Mini cartridges can be connected to any
medium- and high-pressure liquid chromatography
system set to a maximum pressure limit of 45 psi
(3 bar or 300 kPa). Bio-Rad offers two fitting kits for
easy connection of a Bio-Scale Mini cartridge to
medium- or high-pressure chromatography systems.
BioLogic DuoFlow™ Systems
The Bio-Scale Mini cartridge to BioLogic system
fittings kit* includes a 1/4-28 female to male luer
and 1/4-28 female to female luer to connect a
Bio-Scale Mini cartridge to Bio-Rad’s BioLogic
DuoFlow system.
HPLC Systems
The luer to 10-32 adaptor fittings kit* provides
fittings necessary to connect the cartridge to nut
and ferrule type fittings found on most HPLC systems.
8
FPLC Systems
The luer to M6 adaptor fittings kit* provides fittings
necessary to connect the cartridge to the M6 fittings
found on FPLC or related systems.
Section 5
Buffers and Methods
IMAC methods can be run using either native or
denaturing purification protocols. Under native
conditions, proteins are purified using buffers that
help retain the natural folded structure of the target
protein. Under denaturing conditions, strong
chaotropes (typically 6 M urea or guanidine) are
added to the buffers, allowing target proteins to be
purified in their unfolded states. The recommended
buffer compositions and formulations are provided
in the following two tables:
*Fittings kit ordering information can be found within
Section 12 of this manual.
9
Table 4. Buffer Composition
KCl KH2PO4Imidazole Urea
Native lysis/
wash buffer 1 300 mM 50 mM 5 mM N/A
Native wash
buffer 2 300 mM 50 mM 10 mM N/A
Native elution buffer 300 mM 50 mM 250 mM N/A
Denaturing lysis/
wash buffer 1 300 mM 50 mM 5 mM 6M
Denaturing wash
buffer 2 300 mM 50 mM 10 mM 6M
Denaturing elution
buffer 300 mM 50 mM 250 mM 6M
Table 5. Buffer Formulations
KCl KH2PO4Imidazole Urea
Native lysis/wash
buffer 1 22.37 g 6.80 g 0.34 g N/A
Native wash
buffer 2 22.37 g 6.80 g 0.68 g N/A
Native elution buffer 22.37 g 6.80 g 17.02 g N/A
Denaturing lysis/
wash buffer 1 22.37 g 6.80 g 0.34 g 360.36 g
Denaturing wash
buffer 2 22.37 g 6.80 g 0.68 g 360.36 g
Denaturing elution
buffer 22.37 g 6.80 g 17.02 g 360.36 g
10
For all buffer formulations add water to 1 L, adjust
pH to 8.0 with KOH or H
3PO4
, and filter through a
0.2 µM filter.
Native buffers can be stored up to 1 year at
4–22°C; denaturing buffers must be made fresh
and used within 7 days, or frozen in aliquots at
–20°C for later use.
Section 6
Quick Solubility Screening
Protocols
Before choosing a native or denaturing purification
protocol, it is useful to determine the approximate
expression level of a protein, and to determine if the
overexpressed target protein partitions into the
soluble or insoluble fraction. Soluble proteins are
typically purified with the native purification procedure,
while insoluble proteins must be solubilized in
stringent denaturants (urea or guanidine) and are
purified with the denaturing procedure.
11
The following procedure provides a quick screen for
solubility and expression level:
1. Pellet ~ 2 ml of E. coli culture by centrifugation
at 4,000 x g for 10 min at 4°C.
2. Resuspend the pellet in 500 µl of PBS and
sonicate for 60 sec, on ice, in 10 sec pulses.
Remove 50 µl of the sonicate and label as the
"Total" sample. Centrifuge the lysate at
12,000 x g for 10 min at 4°C. Transfer the
supernatant to a clean tube. Remove 50 µl of
the supernatant, and label tube "Soluble".
3. Resuspend the insoluble pellet in 500 µl of 6 M
urea in 1x PBS and sonicate for 60 sec, on ice,
in 10 sec pulses. Centrifuge the lysate at
12,000 x g for 10 min at 4°C. Remove 50 µl of
the supernatant, and label "Insoluble".
4. To each of the 50 µl samples, add 150 µl of
Laemmli buffer, and boil for 5 min at 95°C.
5. Load 10 µl of each sample on an SDS-PAGE
gel.
12
6. Examine the soluble and insoluble fractions for
the target protein. Approximate the expression
level, and determine partitioning of the target
protein.
A partitioning profile of soluble and insoluble target
proteins, with approximate expression levels, can
be seen in Figure 2.
13
~30% expression ~25% expression
Soluble partitioning Insoluble partitioning
Fig. 2. Partioning profiles. For both gels, Precision Plus Protein™
molecular weight markers were loaded in lane 1, followed by the total, soluble,
and insoluble fractions in lanes 2–4 respectively. The first panel depicts a
32 kD target protein, which partitions into the soluble fraction and can be
purified using the native protocol (outlined on page 15). A representative
chromatogram and gel for the purification of this target protein is shown in
Fig. 5 on page 20. The second panel depicts a 24 kD target protein, which
partitions into the insoluble fraction and can be purified using the denaturing
protocol (outlined on page 16).
14
1234 123 4
kD
250
150
100
75
50
37
25
20
15
10
Section 7
Preparation of E. coli
Lysates
For E. coli cultur es expressing medium to high levels of
His-tagged proteins, (≥10% of total protein), 200 ml of
culture will yield sufficient material for a 1 ml cartridge
purification, and 1,000 ml of culture will yield sufficient
material for a 5 ml cartridge purification run. For cultures
expressing protein at low levels (≤10% of total protein),
the culture volumes will need to be determined
empirically for each protein.
Native Lysates
1. Harvest cell pellet by centrifugation at 8,000 x g for
10 min at 4°C.
2. Determine weight of pellet and resuspend in
10 volumes native lysis/wash buffer 1 (200 ml of
culture typically yields 0.8 g of paste, and results in
8 ml of lysate).
15
3. Sonicate the lysate (on ice) 4 times at 1 min intervals.
4. Centrifuge the lysate at 12,000 x g for 20 min at
4°C.
5. Remove the supernatant, and filter through a
0.2 µM filter immediately before applying to the
cartridge.
Denatured Lysates
1. Harvest cell pellet by centrifugation at 8,000 x g for
10 min at 4°C.
2. Determine weight of pellet and resuspend in
10 volumes denaturing lysis/wash buffer 1 (200 ml of
culture typically yields 0.8 g of paste, and results in
8 ml of lysate).
3. Sonicate the lysate 4 times at 1 min intervals.
4. Centrifuge the lysate at 12,000 x g for 20 min at
4°C.
5. Remove the supernatant, and filter through a
0.2 µM filter immediately before applying to the
cartridge.
16
Section 8
Preparing a Cartridge, and
Subsequent Purification
Prepare buffer sets for either the native or denaturing
purification protocols-using a single buffer set
throughout the procedure. To prepare the cartridge
for the procedure, remove the top closure and
connect the cartridge to the chromatography
system. Open the bottom closure and connect the
cartridge outlet to the system. Flush the packing
solution (20% EtOH) from the cartridge by running 2
column volumes (CV) of water at a flow rate of
2 ml/min (1 ml cartridge) or 10 ml/min (5 ml cartridge).
The cartridge is now ready for the purification steps.
Flow rates are given in ml/min and are specific to
the 1 ml cartridge. If a 5 ml cartridge is used for a
procedure, substitute the higher flow rate in the
method (refer to the table below).
17
Table 6. Purification Method Suggestions
1 ml Cartridge 5 ml Cartridge
Step CV Flow Rate Flow Rate
Equilibrate 5 2 ml/min 10 ml/min
Lysate Load 5 to 10 2 ml/min* 10 ml/min*
Wash 1 6 2 ml/min 10 ml/min
Wash 2 6 2 ml/min 10 ml/min
Elute 5 2 ml/min 10 ml/min
Standard methods that are compatible with any
type of chromatography system are listed below. To
maximize binding capacity with large proteins
(>100 kD), for purification at 4°C, or for purifications
under denaturing conditions, the lysate load flow
rate* can be decreased (to 0.5 ml/min for the 1 ml
cartridge and 2 ml/min for the 5 ml). This will have
to be determined empirically for individual proteins.
1. Equilibrate the cartridge with 5 column volumes
(CV) of equilibration/wash buffer 1 at 2 ml/min.
2. Load the sample lysate at 2 ml/min.
3. Wash the cartridge with 6 CV of wash buffer
1 at 2 ml/min.
18
4. Wash the cartridge with 6 CV of wash buffer 2
at 2 ml/min.
5. Elute the purified protein with 10 CV of elution
buffer at 2 ml/min.
6. Prior to quantitation of the protein concentration,
the purified protein should be exchanged into a
non-imidazole buffer (imidazole can absorb at
280 nm). Purified protein from denaturing
purifications should be exchanged into another
buffer through dialysis.
The chromatogram and gel in Figure 5 illustrate a
representative purification of a high-expressing
soluble protein purified using the native buffer set
and method described in Table 6. Note: IMAC
buffers made with potassium salts are more stable
than sodium salt-based buffers. However, potassium
will complex with SDS in Laemmli buffer and
precipitate out of solution. Prior to analyzing IMAC
samples on gels, the samples must be diluted at
least 1:7 with Laemmli buffer to prevent precipitation.
19
20
kD
250
150
100
75
50
37
25
20
15
10
Flowthrough
Native IMAC Purification Profile
Wash-1 Wash-2
Purified
Product
Fig. 5. Native IMAC purification: A 32 kD Nif-3* His-tagged protein was
purified from the soluble fraction using the standard Profinity IMAC native
purification protocol. 2 ml of lysate (2 CV) from a 100 ml E. coli culture was
loaded onto a 1 ml IMAC cartridge. The cartridge was washed with 6 CV of
wash buffer 1, followed by 6 CV of wash buffer 2, and purified protein was
eluted with 5 CV of elution buffer (all at 2 ml/min). The purified product was
>95% pure by densitometric scanning and Quantiy One
®
software analysis.
Lane 1, Precision Plus Protein unstained standards; lane 2, soluble lysate;
lane 3, Flow-through; lane 4, wash 1; lane 5, wash 2; lane 6, purified product.
*Nif-3 construct was kindly provided by R. Stevens, Scripps Institute.
Section 9
Scaling Up
Bio-Scale Mini cartridges are available in 1 ml and
5 ml cartridge formats. The Profinity IMAC resin is
also available in larger amounts, from 25 ml bottles
to bulk quantities, for scaling up methods developed
using the cartridges.
For quick scale-up, two or three cartridges of the
same type can be connected in series; backpressure
will increase with cartridges in series, so care
should be taken to maintain an overall system
pressure ≤45 psi.
In addition, Bio-Rad carries an extensive line of
21
empty chromatography columns from laboratory
scale to process scale. Inquire with your local
Bio-Rad representative or go online to
www.bio-rad.com.
Section 10
Regenerating, Cleaning,
Sanitizing, and Storage
Protein cross-contamination, frit clogging, and
increased backpressure can result from repeating
the number of uses beyond the recommended
number. After repeated use, a cartridge may run
slower or produce higher backpressure, an expected
result due to the very nature of the sample mixture.
The following cleaning and regeneration procedures
may be used, however, it is recommended to
dispose of the cartridge after several uses. To avoid
cross-contamination, it is recommended that single
cartridges are designated for single proteins.
To maintain good flow properties, it is recommended
that the cartridges are cleaned between each use.
22
For the 1 ml cartridges, run the cleaning protocol at
2 ml/min. It is recommended that the 5 ml cartridge
cleaning protocol be run at 5 ml/min.
High Salt/Acid Cleaning
1. Rinse the cartridge with 2 CV water at 2 ml/min.
2. Wash the cartridge with 5 CV 500 mM NaCl,
50 mM Tris, pH 8.0 at 2 ml/min.
3. Wash the cartridge with 5 CV 500 mM NaCl,
100 mM NaOAc, pH 4.5 at 2 ml/min.
4. Rinse the cartridge with 2 CV water at 2 ml/min.
5. Store the cartridge in 20% ETOH at 4°C.
Chaotrope Cleaning
1. Rinse the cartridge with 2 CV water at 2 ml/min.
2. Wash the cartridge with 5 CV 6 M guanidine
HCl at 2 ml/min.
3. Rinse the cartridge with 2 CV water at 2 ml/min.
4. Store the cartridge in 20% ETOH at 4°C.
23
In situations where it is desired to run different
proteins over the same cartridge, a complete
sanitization, stripping, and recharging is
recommended between sample runs. Care should
be taken with the handling and disposal of metal
containing solutions.
1. Clean the cartridge with 10 CV of 1 M NaOH.
2. Rinse the cartridge with 10 CV water.
3. Strip metal ions with 5 CV of 0.1 M EDTA.
4. Rinse the cartridge with 10 CV water.
5. Recharge the cartridge with 5 CV of 0.1 M
nickel sulfate, pH 4.5.
6. Rinse the cartridge with 10 CV water.
7. Store the cartridge in 20% ethanol.
24
Section 11
Troubleshooting Guide
Problem Possible Cause Solution
Cartridge clogging Particulates in samples Filter all samples and buffers
or slow flow rate or buffers through 0.2 µM filter prior to
application
Sample too viscous Add nuclease to lysate to
degrade DNA. Centrifuge
and filter lysate again
No target protein Low level of target Check expression level by
in eluant protein in starting SDS-PAGE
material
Target protein not Check levels of target protein
binding, or eluting in in lysate, flow-through, wash,
wash fractions and eluted fractions. Check
for presence of His-tag with
anti-His antibody
Target protein in His-tag not accessible Reclone His-tag onto opposite
flow through terminus (N or C-terminus)
His-tag not accessible Purify protein under denatur-
ing conditions to expose Histag
Proteolysis & removal Include protease inhibitors in
of tag lysis buffer (or reaction), or
purify in the cold
25
Problem Possible Cause Solution
Precipitation during Binding capacity of Load less sample
purification cartridge exceeded
Protein aggregating Include low levels of
detergent (0.1% Triton
X-100, Tween 20) in
purification. Include glycerol
up to 10%
Protein too concen- Elute with imidazole gradient
trated during elution (10–250 mM) rather than step
elution
Eluted protein is Contaminants co-eluting Elute with imidazole gradient
impure (10–250 mM) rather than step
elution
Contaminants co-eluting Increase the imidazole in the
wash but keep below 40 mM
to increase wash stringency
Target protein is Proteolysis of target Add protease inhibitors to
degraded protein lysate. Purify at 4°C or under
denaturing conditions
26
Section 12
27
Ordering Information
Cartridges
Catalog # Description
732-4610 Bio-Scale Mini Profinity IMAC cartridge, 5 x 1 ml
732-4612 Bio-Scale Mini Profinity IMAC cartridge, 1 x 5 ml
732-4614 Bio-Scale Mini Profinity IMAC cartridge, 5 x 5 ml
For the most up to date list of other cartridge offerings, please visit us
online at www.bio-rad.com/cartridges/
Fittings, Tubing, & Fittings Kits
Catalog # Description
731-8225 1.6 mM Barb to Male Luer
731-8222 1.6 mM Barb to Female Luer
732-0111 Luer to M6 Adaptor Fittings Kit, includes luer to
732-0112 Luer to 10-32 Adaptor Fittings Kit, includes luer
cartridge
cartridge
cartridges
M6 fitting to connect to an FPLC system
to polypropylene/PTFE 10-32 fittings to connect
1 cartridge to an HPLC system
732-0113 Luer to BioLogic System Fittings Kit, includes
1/4-28 female to male luer and 1/4-28 female to
female luer to connect 1 cartridge to the
BioLogic DuoFlow system
• Larger package sizes of media are available for process scale
chromatography. Inquire with your local Bio-Rad representative.
Section 13
References
Joyce AR and Palsson BO,The model organism as a
system: integrating 'omics' data sets, Nat Rev Mol Cell
Biol 7, 198–210 (2006)
Belew M and Porath J, Immobilized metal ion affinity
chromatography. Effect of solute structure, ligand
density and salt concentration on the retention of
peptides, J. Chromatogr 516, 333–354 (1990)
Hochuli E, Large-scale chromatography of
recombinant proteins, J Chromatogr 444, 293–302
(1988)
Maté MJ et al., The crystal structure of the mouse
apoptosis-inducing factor AIF, Nat Struct Biol 9,
442–446 (2002)
28
Porath J et al., Metal chelate affinity chromatography,
29
a new approach to protein fractionation, Nature 258,
598–599 (1975)
Section 14
Legal Notices
FPLC is a trademark of GE Healthcare. Triton is a
trademark of Union Carbide. Tween is a trademark of
ICI Americas, Inc.
Purification and preparation of fusion proteins and
affinity peptides comprising at least two adjacent
histidine residues may require a license under US
patent 5,284,933 and US patent 5,310,663,
including corresponding foreign patents (assignee
Hoffman-La Roche, Inc).
Bio-Rad Laboratories, Inc.
925 Alfred Nobel Dr.
Hercules, CA 94547 USA
510-741-1000
1-800-424-6723
10005455 Rev C
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