BIO RAD | Bio-Scale | Bio-Scale™ Mini Macro-Prep® High Q, High S and DEAE - Bio-Rad

Bio-Scale™ Mini Macro-Prep® High Q, High S and DEAE - Bio-Rad
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Bio-Scale™ Mini
Macro-Prep® High Q,
High S and DEAE
Cartridges, 1 and 5 ml
Instruction Manual
Catalog #
732-4120, 732-4122, 732-4124,
732-4130, 732-4132, 732-4134,
732-4140, 732-4142, 732-4144
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Table of Contents
Section 1 ...Introduction ........................................1
Section 2 Connecting to Bio-Rad’s
Low-Pressure Chromatography
Instruments ........................................5
Section 3 Connection to Other Liquid
Chromatography Systems ................10
3.1
BioLogic DuoFlow™
Systems.................................10
3.2
HPLC Systems ......................11
3.3
FPLC Systems .......................12
Section 4 Preparing a Cartridge for Use...........13
4.1
Sample Preparation ...............14
4.2
General Purification
Protocol .................................15
4.3
Scaling Up the Separation .....16
Section 5 ...Care of the Cartridge........................18
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5.1
Regeneration .........................18
5.2
Cleaning.................................18
5.3
Storage..................................20
Section 6 Technical Assistance ........................20
Section 7 Ordering Information.........................21
Section 8 References .......................................23
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Section 1
Introduction
Bio-Scale Mini cartridges have a patent-pending
double-wall design that provides extra durability and
allows easy, reliable runs with aqueous buffers
commonly used for protein purification. The
polypropylene luer fittings and internal sealing
surfaces ensure leak-free operation at pressures up
to 45 psi. Bio-Scale Mini cartridges are convenient,
disposable, and supplied ready for use. They are
easy to use and prepacked for fast, reproducible
chromatographic separations. Cartridges are available for a variety of chromatographic techniques,
including desalting, ion exchange, affinity, and
mixed-mode chromatography. The design of
Bio-Scale Mini cartridges offers:
•
Ready-to-go convenience; simply equilibrate
the cartridge in the buffer of choice
•
Luer fittings for convenient connection to any
chromatography system or directly to a
Luer-Lok syringe
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Bio-Scale Mini Macro-Prep High Q and S and
Macro-Prep DEAE cartridges are packed with
Macro-Prep® ion exchange media. These media are
based on hydrophilic spherical polymeric beads
designed for the purification of proteins, nucleic
acids, viruses, plasmids, and other macromolecules.
Macro-Prep beads are designed to provide medium
capacity, low backpressure, and high productivity.
Detailed product information is given in Tables 1
and 2. See Ordering Information for a listing of the
complete Bio-Scale Mini cartridge product line.
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Table 1. Bio-Scale Mini Macro-Prep High Q,
High S, and DEAE cartridge specifications.
Sizes
Dimensions
1 ml and 5 ml bed volumes
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
Operational flow rates
1 ml: 1–6 ml/min (240–1460 cm/hr)
5 ml: 1–15 ml/min (50–720 cm/hr)
Fittings:
Female luer inlet;
male luer outlet
Column material
Polypropylene
Frit material
Polyethylene (HDPE)
Shipping conditions
20% ethanol
Storage recommendations
20% ethanol
Autoclavability
Not autoclavable
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Table 2. Macro-Prep High Q & S and
Macro-Prep DEAE specifications.
Properties
High Q
High S
Type of ion exchanger
Total ionic capacity
Strong anion
-N+(CH3)3
400 ± 75 µeq/ml
Strong cation
Weak anion
-SO3-N+(C2H5)2
160 ± 40 µeq/ml 175 ± 75 µeq/ml
Dynamic binding capacity*
≥37 mg BSA/ml
≥49 mg IgG/ml
≥30 mg BSA/ml
Shipping counterion
Chemical stability
1% SDS, 24 hr
6M guanidine-HCl, 24 hr
pH stability
Cl-
Na+
Cl-
Yes
Yes
1–10
Yes
Yes
1–12
Yes
Yes
1–10
Functional group
DEAE
Total organic carbon in ppm, after 24 hours at 22°C
0.1 M HCl
0
4
0.2 M H3PO4
0
13
0.01 M NaOH
28
N/A
0.1 M NaOH
200
31
1.0 M NaOH
337
49
0
0
N/A
26
121
Antimicrobial agent
20% ethanol
20% ethanol
20% ethanol
Regeneration
1–2 M NaCl
1–2 M NaCl
Storage conditions
20% ethanol
20% ethanol
* 10% breakthrough capacity determined in a 1.1 x 20 cm column.
4
1–2 M NaCl
20% ethanol
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Section 2
Connecting to Bio-Rad’s
Low-Pressure
Chromatography
Instruments
Bio-Scale Mini cartridges are ideal for use with
Bio-Rad's BioLogic™ LP chromatography system,
Econo™ gradient pump, the patented* Model EP-1
Econo™ pump, and all low-pressure chromatography instruments. Bio-Scale Mini cartridges can be
conveniently connected directly to the system using
the luer fittings on the cartridge.
1.
Install 1.6 mm inner diameter (ID) tubing in the
pumphead. Adjust the platen pressure screw
(on pumphead) – using a screwdriver or coin,
turn the screw counterclockwise as far as it will
go, then turn clockwise three full turns.
* US patent 5,135,658
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Assemble with fittings and lock rings as shown in
Figure 1.
(Use orange lock rings and medium size barb
fittings with 1.6 mm tubing.)
Platen
pressure
Platen
pressure
screw
screw
Lock-ring
Lock-ring
Tubing
Tubing
Luer
Luerfittimg
fitting
See
detail
See
detail
1
4
7
rm
Ala
C
2
5
8
0
3
6
9
.
Fig. 1. BioLogic LP system setup.
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To maximize gradient accuracy and to apply
samples efficiently, install 1.6 mm ID tubing
from the pump to the MV-6 sample inject valve
(if available). If using the MV-6 sample inject
valve, turn the knob counterclockwise as far as
it will go so it will now correspond to the printed
diagram on the valve (see Figure 2).
SAMPLE LOOP
INJECT
PORT
INJECT
PORT
WASTE
FILL
TO
BIOLOGIC LP
SYSTEM OR ECONO
GRADIENT PUMP
MV-6
INJECT
VALVE
L
FIL
MV-6
TO
INJECT
BIOLOGIC LP
VALVE
SYSTEM OR ECONO
GRADIENT PUMP
SAMPLE LOOP
TO
COLUMN
TO
COLUMN
"FILL" POSITION
"INJECT" POSITION
Fig. 2. Connecting to an MV-6 valve.
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WASTE
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3.
Connect the inlet of the cartridge to the male
luer fitting on the MV-6 sample inject valve (see
Figure 2). If not using the MV-6 sample inject
valve, connect a barb to male luer fitting on the
1.6 mm ID tubing, then connect to the top of
the female luer on the Bio-Scale Mini cartridge.
For optimum performance, a cartridge should
be mounted vertically with the arrow on the
cartridge pointing downward (see Figure 3).
4.
Connect the cartridge outlet to the 1.6 mm ID
tubing leading to the BioLogic LP system optics
module or to the Model EM-1 Econo™ UV
monitor. It is recommended to use the shortest
length (approximately 10 cm) of 1.6 mm ID
tubing. Connect a barb to female luer to the
1.6 mm ID tubing, then connect to the bottom
of the male luer on the Bio-Scale Mini cartridge.
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Fig. 3. Cartridge and fittings.
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Section 3
Connecting to Other Liquid
Chromatography Systems
Bio-Scale Mini cartridges can be connected to any
liquid chromatography system, provided that the
maximum pressure limit (3 bar, 45 psi, or 300 kPa) of
the cartridges is not exceeded. It is recommended
that the system pressure limit be set according to
the cartridge pressure limit. Pressures in excess of
3 bar are usually caused by restrictions in tubing or
detector cells downstream from the cartridge. BioRad offers two fitting kits for easy connection of a
Bio-Scale Mini cartridge to a BioLogic DuoFlow,
HPLC, or FPLC-type system.
3.1 BioLogic DuoFlow Systems
The luer to BioLogic system fittings kit (catalog
#732-0113) includes 1/4-28 female to male luer
and 1/4-28 female to female luer to connect one
Bio-Scale Mini cartridge to the BioLogic DuoFlow
system, see Figure 4.
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Fig. 4. Luer to 1/4-28 adaptor.
3.2 HPLC Systems
The luer to 10-32 adaptor fittings kit (catalog
#732-0112) provides fittings necessary to connect
the Bio-Scale Mini cartridge to nut and ferrule type
fittings found on most HPLC systems. Alternatively,
the cartridge can be connected to HPLC systems
via a low dead-volume 1/16 inch union with a new
piece of stainless-steel tubing attached to the
union. Simply slip a short length of the 0.8 mm ID
tubing over the 1/16 inch OD stainless-steel tubing
to a distance of 1 cm.
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3.3 FPLC Systems
The luer to M6 adaptor fittings kit (catalog #732-0111)
provides fittings necessary to connect the Bio-Scale
Mini cartridge to the M6 fittings found on FPLC or
related systems. Alternatively, connection can be made
by using one GE Healthcare Union luer lock female
to M6 female fitting (GE 18-1027-12) and one
female luer lock to M6 male fitting (Upchurch P-686
or GE 18-1027-62). To prevent tubing or cartridge
failure, do not exceed the maximum recommended
flow rate of the cartridge.
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Section 4
Preparing a Cartridge
For Use
Bio-Scale Mini Macro-Prep High Q, Macro-Prep
High S, and Macro-Prep DEAE cartridges contain
20% ethanol (v/v) as the storage solution. The fully
hydrated support is ready to use after equilibrating
the cartridge in the buffer of choice. To perform
buffer exchange, connect the cartridge to a liquid
chromatography system or peristaltic pump and
condition it as instructed below:
1.
Set pump flow rate to 3.0 ml/min (731 cm/hr)
for the 1 ml cartridge or 6.0 ml/min (288 cm/hr)
for the 5 ml cartridge.
2.
Wash the cartridge with degassed low-salt
buffer for 2 min.
3.
Wash the cartridge with degassed high-salt
buffer for 5 min.
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4.
Equilibrate the cartridge with low-salt buffer for
5 min.
5.
Reduce the flow rate to that which will be used
in the purification protocol.
4.1 Sample Preparation
Proper pH and ionic strength are necessary for
consistent and reproducible results. Sample can be
exchanged into the starting buffer or diluted to the
starting buffer's concentration. This can be
achieved by diluting the sample to the ionic strength
of the starting buffer, dialyzing against the starting
buffer, or exchanging it into the starting buffer.
Buffer exchange can be accomplished using a
Bio-Scale Mini P-6 cartridge, Bio-Spin® 6 or
Bio-Spin 30 column, Econo-Pac® 10DG desalting
column, or Bio-Gel® P-6DG gel filtration gel (see
Table 3). The choice of product will depend on
sample volume. All samples should be filtered
through a 0.45 µm filter prior to cartridge application.
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Table 3. Products for buffer exchange.
Sample
Volume
Recommended
Product
Use
Catalog #
50–100 µl
Bio-Spin 6 column
Desalting proteins
≥ 6 kD
732-6002
50–100 µl
Bio-Spin 30 column
Desalting proteins
≥ 30 kD
732-6006
100 µl–3 ml
Bio-Scale Mini P-6
cartridge
Desalting proteins
≥ 6 kD
732-4502
Up to 3 ml
Econo-Pac 10DG
desalting column
Desalting proteins
≥ 6 kD
732-2010
Unlimited
Bio-Gel P-6DG gel
Desalting proteins
≥ 6 kD
150-0738
4.2 General Purification Protocol
Ion exchange chromatography is usually performed
using increasing salt gradients or pH gradients to
elute the sample components. For best results and
to increase cartridge life, samples and buffers should
be degassed and filtered through a 0.45 µm filter.
Common buffers for cation and anion exchange
chromatography are listed in Table 4. An appropriate
starting point for purifying samples is a linear
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gradient from 0.0 to 0.4 M NaCl spanning 1 to 20
column volumes at 120 cm/hr, or 0.5 ml/min for the
1 ml cartridge and 2.5 ml/min for the 5 ml cartridge.
The separation can be optimized by changing the
gradient profile. At the end of each run, the cartridge
can be regenerated with 1.0 M NaCl followed by
starting buffer. Return to the desired flow rate and
proceed with the next separation.
4.3 Scaling Up the Separation
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 pressures ≤45 psi.
Bio-Scale Mini cartridges are available in 1 ml and
5 ml cartridge formats. The Macro-Prep High Q,
High S, and DEAE ion exchange media are also
available in larger amounts, from 25 ml bottles to
bulk quantities, for scaling up methods developed
using the cartridges. Macro-Prep High Q, High S,
and DEAE media are fully supported with Regulatory
Support Files. In addition, Bio-Rad carries an
extensive line of empty chromatography columns
from laboratory scale to process scale.
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Table 4. Common buffers for ion exchange
chromatography.
Type of Buffering
Buffer Range
Cation Exchanger
Acetic acid
Citric acid
HEPES
Lactic acid
MES
MOPS
Phosphate
PIPES
Pivalic acid
TES
Tricine
Anion Exchanger
Bicine
Bis-Tris
Diethanolamine
Diethylamine
L-Histidine
Imidazole
Pyridine
Tricine
Triethanolamine
Tris
4.8–5.2
4.2–5.2
7.6–8.2
3.6–4.3
5.5–6.7
6.5–7.9
6.7–7.6
6.1–7.5
4.7–5.4
7.2–7.8
7.8–8.9
7.6–9.0
5.8–7.2
8.4–8.8
9.5–11.5
5.5–6.0
6.6–7.1
4.9–5.6
7.8–8.9
7.3–8.0
7.5–8.0
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Section 5
Care of the Cartridge
5.1 Regeneration
After each use, the cartridge should be regenerated
with the appropriate salt, in most cases 1–2 M NaCl
in the presence of buffer. Wash with 2 to 4 column
volumes of the buffered high salt solution. This
reduces the potential for protein precipitation when
selecting acid as a cleaning agent.
5.2 Cleaning
After repeated use, an ion exchange cartridge may
require thorough cleaning and regeneration to
remove bound contaminants. Acceptable cleaningin-place (CIP) reagents include 1% acetic acid/1%
phosphoric acid with 0.4 M NaCl, up to 30% acetic
acid, 1% Triton X-100, up to 70% ethanol or 30%
isopropyl alcohol, 8 M urea, and 6 M guanidine-HCl.
Any of these agents can be combined in an
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appropriate cleaning protocol. As a general guide, we
recommend the following:
1.
Use high salt buffer for regeneration, as above.
2.
For aggregated or precipitated proteins, or
when dirty feed stock (e.g., crude lysate) has
been used, wash with 3–5 column volumes of
6 M guanidine-HCl or 8 M urea at 100 cm/hr.
3.
For lipids or hydrophobically bound contaminants,
wash with 0.1% Triton X-100 or 20–70%
ethanol or isopropyl alcohol, or 1–30% acetic
acid. Use 3–5 column volumes at 100 cm/hr.
4.
Remove additional contaminants with 0.4 M
NaCl in 1% acetic acid/1% phosphoric acid.
Use 3–5 column volumes at 100 cm/hr.
5.
If the cartridge is to be used again immediately,
wash with 2 column volumes of deionized
water and 4–5 column volumes of starting
buffer at 100 cm/hr. Check the conductivity
and pH of the effluent to verify that the column
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is equilibrated in the starting buffer before
loading the sample.
5.3 Storage
After washing the cartridges with deionized water,
Bio-Scale Mini ion exchange cartridges should be
purged and stored with PBS, containing 0.05%
NaN3, or in 20% (v/v) ethanol solution, and capped
for extended storage.
Section 6
Technical Assistance
For additional information and technical assistance,
contact your local Bio-Rad representative as listed on
the back cover of our catalog, or in the USA call
Technical Support at 1-800-4BIORAD.
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Section 7
Ordering Information
Bio-Scale Mini Cartridges*
Description
5 x 1 ml 1 x 5 ml 5 x 5 ml
732-4100 731-4102 731-4104
UNOsphere™ Q Support
UNOsphere S Support
732-4110 731-4112 731-4114
Macro-Prep™ High Q Support 732-4120 732-4122 732-4124
Macro-Prep High S Support
732-4130 732-4132 732-4134
Macro-Prep DEAE Support
732-4140 732-4142 732-4144
Bio-Gel P-6 Support
—
732-4502 732-4504
—
Affi-PRep® Protein A Support 732-4600 732-4602
732-4610 732-4612 732-4614
Profinity™ IMAC Support
—
732-4632 732-4634
Affi-Gel® DEAE Blue Support
Affi-Gel Blue Support
—
742-4642 732-4644
* Visit www.bio-rad.com/cartridges/ for current information on
prepacked cartridges.
• Larger package sizes of media are available for process-scale
chromatography. Inquire with your local Bio-Rad representative.
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Catalog #
Fittings Kits
Description
732-0111
Luer to M6 Adaptor Fittings Kit, includes luer to
M6 fitting to connect to an FPLC system
732-0112
Luer to 10-32 Adaptor Fittings Kit, includes luer
to polypropylene/PTFE 10-32 fittings to connect
1 cartridge to an HPLC system
732-0113
Luer 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
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Section 8
References
1. Gagnon P, Avoiding Instrument-associated
Aberrations in Purification Scale-up and Scaledown, BioPharm 10, 42–45 (1997)
2. Harris ELV and Angal S, Protein Purification
Methods: A Practical Approach, IRL Press,
Oxford (1989)
3. Scopes RK, Protein Purification: Principles and
Practice (Second Edition), Springer-Verlag, New
York (1987)
4. Snyder LR and Kirkland JJ, Introduction to
Modern Liquid Chromatography (Second Edition),
Wiley, New York (1979)
FPLC is a trademark of GE Healthcare. Luer-Lok is a trademark of
Becton, Dickinson and Co. Triton is a trademark of Union Carbide.
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Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
510-741-1000
1-800-424-6723
10006400
Rev C
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