Bio-Rad Bio-Scale Mini Affi-Gel Blue Cartridges User Manual

Bio-Scale™Mini Affi-Gel

®

Blue and DEAE Affi-Gel
Blue Cartridges, 5 ml

Instruction Manual

Catalog #
732-4642, 732-4644
732-4632, 732-4634

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 Connecting to 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 ...............16

4.2 General Purification

Protocol .................................18

4.3 Scaling Up the Separation .....18

Section 5 Specific Purification Protocols ..........19

5.1 Purification of IgG from
Serum or Ascites with
the Bio-Scale Mini DEAE Affi-Gel

Blue Cartridge........................19

5.2 Purification of Serum
Proteins with the
Bio-Scale Mini DEAE Affi-Gel

Blue Cartridge........................20

5.3 Removal of Albumin from
Serum with the Bio-Scale Mini

Affi-Gel Blue Cartridge ...........21

5.4 Purification of Enzymes
with the Bio-Scale Mini Blue

Affi-Gel Cartridge ...................22

Section 6...Care of the Cartridge........................23

6.1 Cleaning the Cartridge ...........23

6.2 Storage..................................24

Section 7 Technical Assistance ........................25

Section 8 Ordering Information.........................26

Section 9 References .......................................28

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-use 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

1

Bio-Scale Mini Affi-Gel Blue and DEAE Affi-Gel Blue
cartridges are packed with Affi-Gel

®

affinity
chromatography gels. These gels are hydrophilic
spherical crosslinked agarose beads designed for
the purification of proteins or the removal of
high-abundance proteins from cell lysates or serum.
Request bulletin 1107 for other applications. Affi-Gel
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.

2

Table 1. Bio-Scale Mini Affi-Gel Blue and
DEAE Affi-Gel Blue cartridge specifications.

Size 5 ml bed volume

Dimensions 40 mm length x 12.6 mm inner diameter

Maximum pressure tolerance 10 psi (680 mbar. 0.36 mPa)*

Operational flow rates 0.5–2.0 ml/min (25–100 cm/hr)

Fittings: Female luer fitting inlet;

male luer fitting outlet

Column material Polypropylene

Frit material Polyethylene (HDPE)

Shipping conditions 0.02 M sodium phosphate, pH 7.4,

0.05% NaN

3

Storage recommendations 0.02 M sodium phosphate, pH 7.4,

0.05% NaN

3

Autoclavability Not autoclavable

*Although the pressure limitation of the cartridge is 45 psi, the agarose gel
tends to compress above 10 psi (generally 3.0 ml/min).

3

Table 2. Affi-Gel Blue and DEAE Affi-Gel
Blue specifications.

AAffffii--GGeell BBlluuee DDEEAAEE AAffffii--GGeell BBlluuee

Type Dye affinity Dye affinity/weak anion

Functional group Cibracron Blue F3GA Cibracron Blue F3GA

and – N(C

2H5)2

Serum capacity 0.3–1.0 ml 0.3–1.0 ml

Maximum flow rate 3.0 ml/min 3.0 ml/min

Operating pH range 2–10 2–10

pH stability 1–10 1–12

Chemical compatibility See Table 3.

4

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. Assemble
with fittings and lock rings as shown in Figure 1.

*US Patent 5,135,658

5

(Use orange lock rings and medium size barb
fittings with 1.6 mm tubing.)

Fig. 1. BioLogic LP system setup.

6

Platen pressure

screw

See detail

Lock-ring

Tubing

Luer
fitting

Platen pressure

screw

See detail

23

1

9

456

7 8

0

C .

Alarm

Lock-ring

Tubing

Luer fittimg

2. 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).

Fig. 2. Connecting to an MV-6 valve.

7

SAMPLE LOOP

INJECT
PORT

MV-6

BIOLOGIC LP

SYSTEM OR ECONO

GRADIENT PUMP

TO

INJECT

VALVE

FILL

TO

COLUMN

WASTE

SYSTEM OR ECONO

GRADIENT PUMP

BIOLOGIC LP

SAMPLE LOOP

INJECT
PORT

FILL

MV-6

INJECT

TO

VALVE

"INJECT" POSITION"FILL" POSITION

TO

COLUMN

WASTE

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.

8

Fig. 3. Cartridge and fittings.

9

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

3.4 bar are usually caused by restrictions in
tubing or detector cells downstream from the
cartridge. Bio-Rad 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.

10

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.

11

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.

12

Section 4
Preparing a Cartridge For
Use

Bio-Scale Mini Affi-Gel Blue and DEAE Affi-Gel Blue
cartridges contain 0.02 M sodium phosphate buffer
pH 7.4 and 0.05% sodium azide 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:

Both Bio-Scale Mini affinity cartridges are packed
with sterile buffer containing sodium azide and are
shipped in a fully hydrated state to maximize shelf
life. New cartridges should always be prepared
according to the instructions in this section. Refer to
Table 3 for the formulation of the buffers discussed
in this section. If air is accidently introduced to a
cartridge, it can be easily removed following these

13

same instructions. After connecting the cartridge to
a liquid chromatography system, prepare it as follows
using Table 3 as a guide.

1. Set pump flow rate to 1.0 ml/min.

2. Wash the cartridge with degassed regeneration
buffer (G) for 10 minutes at 1.0 ml/min.

3. Wash the cartridge with degassed elution
buffer (D or E) for 10 minutes at 2.0 ml/min.

4. Wash the cartridge with degassed application
buffer (A, B, or C) for 10 minutes at 2.0 ml/min.

5. Equilibrate the cartridge with degassed
application buffer for 2 minutes at 1.0 ml/min.

14

Table 3. Buffer formulations.

Application Buffers

A. 0.028 M NaCl, 0.020 M Tris-HCl, pH 8.0

B. 0.020 M K

2

HPO4, pH 8.0

C. 0.020 Na

2

HPO4, pH 7.1

Elution Buffers

D. 0.4 M K

2

HPO4, pH 8.0

E. 1.4 M NaCl, 0.020 M Tris-HCl, pH 8.0

F. 1.4 M NaCl, 0.020 M Na

2

HPO4, pH 7.1

Regeneration Buffers

G. 1.4 M NaCl, 0.10 M acetic acid, pH 3.0, 40% v/v isopropyl

alcohol

H. 1.5 M sodium thiocyanate in application buffer A, B, or C

I. 2.0 M guanidine-HCl in application buffer A, B, or C

15

Table 4. Recommended application buffer
for specific samples.

Target Bio-Scale Mini

Sample Compound Cartridge Buffer

Rabbit, rat, goat, IgG DEAE Blue A
or sheep serum

Human serum IgG DEAE Blue B

Serum Albumin Blue C

Physiological fluids Enzymes Blue Varies

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-6 DG gel filtration gel (seeTable 5). The

choice of product will depend on sample volume. All

16

samples should be filtered through a 0.45 µm filter
prior to cartridge application.

Table 5. Products for buffer exchange.

Sample Recommended
Volume Product Use Catalog #

50–100 µl Bio-Spin 6 column Desalting proteins 732-6002

≥ 6 kD

50–100 µl Bio-Spin 30 column Desalting proteins 732-6006

≥ 30 kD

100µl–3 ml Bio-Scale Mini P-6 Desalting proteins 732-4502

cartridge ≥ 6 kD

Up to 3 ml Econo-Pac 10DG Desalting proteins 732-2010

desalting column ≥ 6 kD

Unlimited Bio-Gel P-6DG gel Desalting proteins 150-0738

≥ 6 kD

17

4.2 General Purification Protocol

Affinity chromatography can be performed using
isocratic elution or by increasing salt or pH gradients
to fractionate the sample components. For best
results, and increased cartridge life, samples, and
buffers should be degassed and filtered through a

0.45 µm filter. Table 4 references selected buffers

for some specific applications. Buffer formulations
are listed in Table 3.

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
≤ 10 psi. Bio-Scale Mini Affi-Gel Blue and DEAE
Affi-Gel Blue cartridges are available in 5 ml format.
The Affi-Gel Blue and DEAE Affi-Gel Blue media are
also available in 100 ml bottles for scaling up
methods developed using the cartridges. In addition,
Bio-Rad carries an extensive line of empty
chromatography columns from laboratory scale to
process scale.

18

Section 5
Specific Purification
Protocols

Several specific application protocols have been
developed using dye affinity supports. Examples
follow.

5.1 Purification of IgG from Serum or

Ascites With the Bio-Scale Mini DEAE
Affi-Gel Blue Cartridge

IgG can be isolated from serum or ascite samples
using the Bio-Scale Mini DEAE Affi-Gel Blue
cartridge. The resulting purified IgG fraction may
contain a residual amount of transferrin.

1. Equilibrate the cartridge in application buffer (A
or B).

2. Apply the prepared sample to the cartridge.

19

3. Elute the IgG with 10-20 ml application buffer.
Smaller volume fractions should be collected
for more precise collection of the IgG fraction.

4. Optional: Most of the bound albumin can be
eluted by washing the cartridge with 10–20 ml
of elution buffer (D or E).

5. Regenerate the cartridge as recommended in
Section 6.1, Cleaning the Cartridge.

5.2 Purification of Serum Proteins With the

Bio-Scale Mini DEAE Affi-Gel Blue Cartridge

Serum proteins have been purified using linear
gradients on DEAE Affi-Gel Blue gel.1 For gradient
separations on the Bio-Scale Mini DEAE blue
cartridge, an appropriate starting point is a linear
gradient from application buffer (B) to elution buffer
(D) over 60 minutes. The remaining bound proteins
are eluted with elution buffer (E). The flow rate is
usually set between 0.5 and 2.0 ml/min. The
separation can then be optimized by changing the

20

flow rate and gradient profile. Request bulletin 1092
for further information on the purification of serum
proteins using DEAE Affi-Gel blue gel.

5.3 Removal of Albumin from Serum With

the Bio-Scale Mini Affi-Gel Blue Cartridge

The Bio-Scale Mini Affi-Gel Blue cartridge can
provide a simple first step in the purification of many
serum proteins by removing the major serum
component, albumin.

1. Equilibrate the cartridge in application buffer (C).

2. Apply the prepared sample to the cartridge.

3. Wash the cartridge with 10–15 ml of application
buffer (C). The effluent from this step contains
the serum proteins minus most of the albumin.

4. Optional: Most of the bound albumin can be
eluted by washing the cartridge with 10-20 ml
of elution buffer (F).

21

5. Regenerate the cartridge as recommended in
Section 6.1, Cleaning the Cartridge.

5.4 Purification of Enzymes With the

Bio-Scale Mini Affi-Gel Blue Cartridge

The Bio-Scale Mini Affi-Gel Blue cartridge can be
used to purify a number of enzymes, especially
kinases, dehydrogenases, and other nucleotide­dependent enzymes.

1. Equilibrate the cartridge in application buffer.
The application buffer will vary depending upon
the enzyme to be purified. In general, the
application buffer should be low ionic strength,

0.05 M or less, with pH between 6.0–8.5.

2. Apply the prepared sample to the cartridge.

3. Wash the cartridge with 10 ml application
buffer.

4. Check the effluent for enzyme activity. If the
enzyme of interest is bound by the cartridge,

22

proceed to step 5. If the enzyme is not bound,
alter the application conditions: change the pH,
change the buffer, or decrease ionic strength.

5. The enzyme can be eluted with a salt gradient
2 or with a competitive eluant such as a
cofactor. Examples of salt gradients include

0.05 to 1.5 M NaCl

3, 4, 5

or 0.0 to 3.0 M KCl.

6,

7, 8

Request technical bulletin 1107 for more
information on elution buffers for enzyme
purification.

6. Regenerate the cartridge as recommended in
Section 6.1, Cleaning the Cartridge.

Section 6
Care of the Cartridge

6.1 Cleaning the Cartridge

After each use, both Bio-Scale Mini dye affinity cartridges
require thorough cleaning and regeneration to
remove bound contaminants. Referring to Table 4

23

for buffers, bound contaminants may be removed
by following the procedure below:

1. Set the pump flow rate to 2.0 ml/min.

2. Wash the cartridge with 10 ml of elution buffer
(E or F).

3 Wash with 10 ml regeneration buffer (H or I).

4. Wash with 20 ml application buffer (A, B, or C).

5. Reduce the flow to 1.0 ml/min.

6. Continue with sample application.

6.2 Storage

Bio-Scale Mini dye affinity cartridges should be
stored at 4 °C in 0.020 M sodium phosphate buffer
containing 0.05% sodium azide. Perform steps 1–3
in the Cleaning the Cartridge section, then wash
with storage buffer.

24

Section 7
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 US, call
Technical Support at 1-800-4BIORAD.

25

Section 8
Ordering Information

Bio-Scale Mini Cartridges*
Description 5 x 1 ml 1 x 5 ml 5 x 5 ml

UNOsphere

™

Q Support 732-4100 731-4102 731-4104
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 —

Profinity

™

IMAC Support 732-4610 732-4612 732-4614
Affi-Gel Blue Support – 732-4642 732-4644
DEAE Affi-Gel Blue Support – 732-4632 732-4634
* 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.

26

Catalog # Description

27

Fittings Kits

732-0111 Luer to M6 Adaptor Fittings Kit, includes luer to

732-0112 Luer to 10-32 Adaptor Fittings Kit, includes luer

732-0113 Luer to BioLogic System Fittings Kit, includes

M6 fitting to connect to an FPLC system

to polypropylene/PTFE 10-32 fittings to connect
1 cartridge to an HPLC system

1/4-28 female to male luer and 1/4-28 female to
female luer to connect 1 cartridge to the
BioLogic DuoFlow system

28

Section 9
References

1. Williams CA, Methods in Immunology and

Immunochemistry, Williams CA and Chase MW
(eds), Academic Press, New York (1967)

2. Wilson JE, Applications of blue dextran and

Cibacron Blue F3GA in purification and structural
studies of nucleotide-requiring enzymes, Biochem
Biophys Res Commun 72, 816–823 (1976)

3. Keith JM et al., Purification and characterization

of the messenger ribonucleic acid capping
enzyme GTP:RNA guanylyltransferase from
wheat germ, Biochemistry 21, 327–333 (1982)

4. Tomasselli AG and Noda LH, Mitochondrial

ATP:AMP phosphotransferase from beef heart:
purification and properties, Eur J Biochem 103,
481–491 (1980)

5. Sharma RK et al., Purification and properties of

bovine brain calmodulin-dependent cyclic
nucleotide phosphodiesterase, J Biol Chem 255,
5916–23 (1980)

29

6. Chetsanga CJ, Purification and characterization

of Escherichia coli formamidopyrimidine-DNA
glycosylase that excises damaged
7-methylguanine from deoxyribonucleic acid,
Biochemistry 20, 5201-5207 (1981)

7. Kattchee PA, Enzymatic assay of

5-methyl-L-tetrahydrofolate, Anal Biochem 118,
85–90 (1981)

8. Bisson LF and Thorner JJ, Thymidylate

synthetase from Saccharomyces cerevisiae.
Purification and enzymic properties, J Biol Chem
256, 12456–12462 (1981)

Cibacron is a trademark of Ciba-Geigy Corporation. FPLC is a
trademark of GE Healthcare. Luer-Lok is a trademark of Becton,
Dickinson and Co. Triton is a trademark of Union Carbide.

Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
510-741-1000
1-800-424-6723

10006402 Rev C