BIO RAD Bio-Scale Bedienungsanleitung
Bio-Scale™Mini
Profinity eXact™
Cartridges, 1 and 5 ml
Instruction Manual
Catalog #
732-4646
732-4648
10011164A.qxp 11/19/2007 9:54 AM Page I
Table of Contents
Section 1...Introduction ........................................1
Section 2 Product Information............................2
Section 3 Connection to Low-Pressure
Chromatography Systems ..................7
Section 4 Connection to Medium- and High-
Pressure Chromatography Systems .11
4.1 BioLogic DuoFlow™
Systems .................................11
4.2 HPLC Systems .......................12
4.3 FPLC Systems........................13
Section 5...Buffers and Methods........................14
Section 6 Quick Solubility Screening
Protocol............................................16
Section 7 Preparation of E. coli Lysate .............19
Section 8 Cartridge Preparation and
Purification Protocol .........................20
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
510-741-1000
1-800-424-6723
10011164 Rev B
10011164A.qxp 11/19/2007 9:54 AM Page II
Section 1
Introduction
Bio-Scale Mini cartridges are convenient, disposable,
prepacked chromatography cartridges. Patented*
column design assures leak-free operation with any
low pressure chromatography system. Bio-Scale
Mini cartridges are available for a variety of
chromatographic techniques such as desalting, ion
exchange, hydrophobic interaction, and affinity
chromatography.
Bio-Scale Mini Profinity eXact cartridges are packed
with Profinity eXact (EXact Affinity Cleavage Technology)
purification resin. This agarose-based affinity
chromatography resin utilizes an immobilized, modified
protease that selectively binds the fusion protein
and cleaves the affinity tag on-column under
controlled conditions, releasing the purified target
protein which contains only its native amino acid
sequence. This innovative resin technology improves
the efficiency of recombinant protein purification, and
is the only affinity chromatography platform that
completes the purification and tag removal process in
a single step.
*U.S. patent 7,208,087 1
Section 9 Scaling Up........................................23
Section 10 Regenerating, Cleaning,
and Storage .....................................23
Section 11 Troubleshooting Guide......................26
Section 12 Ordering Information.........................28
Section 13 References .......................................29
Section 14 Legal Notices ...................................29
10011164A.qxp 11/19/2007 9:54 AM Page IV
Table 1. Bio-Scale Mini Profinity eXact
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
Maximum flow rate 1 ml: 3 ml/min (720 cm/hr)
5 ml: 10 ml/min (480 cm/hr)
Fittings Female luer inlet and
male luer outlet
Column material Polypropylene
Frit material Polyethylene (HDPE)
Shipping conditions 100 mM sodium phosphate,
0.02% sodium azide, pH 7.2
Storage recommendations 100 mM soidum phosphate,
0.02% sodium azide, pH 7.2
Autoclavability Not autoclavable
3
Section 2
Product Information
Bio-Scale Mini cartridges are prepacked
chromatography cartridges supplied ready for use in
1 ml and 5 ml sizes. Bio-Scale Mini 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.
See Ordering Information, Section 12, for a listing of
the complete Bio-Scale Mini cartridge product line.
2
10011164A.qxp 11/19/2007 9:54 AM Page 2
Table 3. Buffer and chemical compatibilities for
Profinity eXact cartridges.
Reagent
Group Description Stability
Triggering Ions F-, Cl-, N3-, NO2-, CO2-Do not use in lysis and
wash buffers.
See Table 5 for use as
trigger in elution buffer.
Salts Sodium Acetate £3M
NaCl or KCl Do not use in lysis and
wash buffers.
Buffers Tris-HCl Substitute Tris-acetate
or Tris-phosphate.
Acids HCl Do not use. Substitute
acetic or phosphoric
acid.
Detergents Non-ionic £5% (w/v)
Zwitterionic £5% (w/v)
Ionic Do not use.
Protease PMSF, CalBioChem cocktail,
inhibitors Roche Protease Inhibitor 1X
cOmplete mini tablet
BD Biosciences cocktail 2X
Lysis Solutions Lysis & Extractrion Reagent 1X
(Bio-Rad), B-PER phosphate
(Pierce), BugBuster (EMD),
FastBreak Cell Lysis (Promega)
ReadyPreps Lysis (Epicentre), Do not use.
CelLytic Express (Sigma)
5
Table 2. Profinity eXact resin specifications.
Functional ligand Mutant subtilisin
Base bead Superflow 6% agarose
Particle size range 60–160 µm
Recommended linear flow rate <1,000 cm/hr at 25°C
Maximum operating pressure 45 psi
Chemical compatibility See Table 3
Storage 4°C
Shelf life in storage buffer >1 year at 4°C
Operational temperature 4–40°C
4
10011164A.qxp 11/19/2007 9:54 AM Page 4
Section 3
Connection to
Low-Pressure
Chromatography Systems
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
7
Reagent
Group Description Stability
Denaturants Guanidine-HCl Do not use in lysis and
wash buffers.
Urea Up to 2 M with no drop
in binding capacity. At
4 M there may be some
loss of binding capacity.
At 8 M, binding capacity
and target protein purity
will be reduced.
Other additives CaCl2£5 mM when used with
MES, MOPS, or PIPES
buffers. Do not use with
phosphate buffers.
MgCl2£5 mM. Do not use with
fluoride containing
buffers. Use NaN3as an
alternate triggering ion.
For a complete list of chemical compatibility, refer to the Profinity eXact
Purification System instruction manual.
.
6
10011164A.qxp 11/19/2007 9:54 AM Page 6
Fig. 2. Connecting to an MV-6 valve.
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).
TO
COLUMN
TO
BIOLOGIC LP
SYSTEM OR ECONO
GRADIENT PUMP
TO
BIOLOGIC LP
SYSTEM OR ECONO
GRADIENT PUMP
MV-6
INJECT
VALVE
SAMPLE LOOP
WASTE
INJECT
PORT
FILL
TO
COLUMN
MV-6
INJECT
VALVE
SAMPLE LOOP
WASTE
INJECT
PORT
FILL
"INJECT" POSITION"FILL" POSITION
9
Fig. 1. BioLogic LP system setup. (Use orange lock rings
and medium size barb fittings with 1.6 mm tubing.)
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 corresponds to the printed diagram
on the valve (see Figure 2).
1
Alarm
23
456
7 8
0
9
C .
Platen pressure
screw
Lock-ring
Tubing
Luer fittimg
See detail
8
10011164A.qxp 11/19/2007 9:54 AM Page 8
Section 4
Connection to Medium
and High-Pressure
Chromatography Systems
Bio-Scale Mini cartridges can be connected to any
liquid chromatography system, provided that the
system does not exceed the maximum pressure
limit of the cartridges (3 bar, 45 psi or 300 kPa). 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. Bio-Rad offers two fitting kits for easy
connection of a Bio-Scale Mini cartridge to a
BioLogic DuoFlow™, HPLC-, or FPLC-type system.
4.1 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)
11
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.
Fig. 3. Cartridge and fittings. Luer fittings and column: a
cartridge should be mounted vertically with the arrow on the
cartridge pointing downward.
10
10011164A.qxp 11/19/2007 9:54 AM Page 10
4.3 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 luerlock
female to M6 female fitting (GE 18-1027-12) and
one Upchurch P-686, female slip luer to male M6
fitting or GE 18-1027-62, Union luerlock female to
M6 male fitting. To prevent tubing or cartridge failure,
do not exceed the maximum recommended flow
rate of the cartridge.
* Fittings kit ordering information can be found within
the Ordering Information section of this manual.
13
Fig. 4. Luer to 1/4-28 adaptor.
4.2 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.
12
10011164A.qxp 11/19/2007 9:54 AM Page 12
Table 4. Buffer composition.
Bind/wash buffer* 100 mM sodium phosphate, pH 7.2
Elution buffer* 100 mM sodium phosphate,
100 mM sodium fluoride, pH 7.2
Regeneration 100 mM phosphoric acid
Storage 0.02% sodium azide,
100 mM sodium phosphate, pH 7.2
* Add urea to the bind/wash buffer and elution buffer in order to purify proteins
under denaturing conditions.
Table 5. Triggering anions.
Anion Compound Fast Cleavage Slow Cleavage
F-NaF, KF 100 mM 5 mM
N3-NaN310 mM 1 mM
NO2-NaNO25 mM 1 mM
CO2-NaHCO21000 mM 25 mM
Cl-NaCl, KCl >1000 mM 75 mM
15
Section 5
Buffers and Methods
Bio-Scale Mini Profinity eXact cartridges 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, a
strong chaotrope (e.g. urea) is included in the
bind/wash buffer and elution buffer, allowing target
proteins to be purified in an unfolded or partially
folded state. The recommended buffer compositions
and triggering anions are provided in the following
tables. Note that some loss in binding capacity may
be observed when buffers used contain greater
than 2 M urea. Additionally, urea concentrations
greater than 4 M may result in decreased target
protein purity.
14
10011164A.qxp 11/19/2007 9:54 AM Page 14
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