BIO RAD Bio-Scale User manual

Bio-Scale™Mini
Profinity eXact™
Cartridges, 1 and 5 ml
Instruction Manual
Catalog #
732-4646
732-4648
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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
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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
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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
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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
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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
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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
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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
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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
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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
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supernatant to a clean tube. Remove 50 µl of
the supernatant, and label the tube "Soluble".
3. Resuspend the insoluble pellet in 500 µl of
bind/wash buffer containing 4 M urea.
Centrifuge the lysate at 16,000 x g for 5 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 heat for 5 min at 95°C.
5. Load 10 µl of each sample on an SDS-PAGE
gel.
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 a soluble target protein can be
seen in Figure 5.
17
Section 6
Quick Solubility Screening
Protocol
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 can be solubilized in urea
and purified with the denaturing procedure.
The following procedure provides a quick screen for
solubility and expression level:
1. Pellet ~ 2 ml of E. coli culture by centrifugation
at 16,000 x g for 1 min at 4°C.
2. Resuspend the pellet in 500 µl of bind/wash
buffer 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
16,000 x g for 5 min at 4°C. Transfer the
16
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Section 7
Preparation of E. coli Lysate
For E. coli cultures expressing medium to high levels of
Profinity eXact-tagged proteins, (≥10% of total protein),
50 ml of culture will yield sufficient material for a 1 ml
cartridge purification, and 250 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.
Lysate Preparation
1. Harvest cells by centrifugation.
2. Determine weight of cell pellet and resuspend in
10 volumes of bind/wash buffer (50 ml of culture
typically yields 0.5 g of paste, resulting in
approximately 5 ml of lysate).
3. Sonicate the lysate (on ice) for 3 minutes.
4. Centrifuge the lysate at ≥16,000 x g for 30 min at
4°C.
19
Fig. 5. Partioning profile. 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 gel depicts
Profinity eXact-tagged Maltose Binding Protein, which partitions
into the soluble fraction and can be purified using the native
purification protocol. A representative chromatogram and gel for
the purification of this target protein is shown in Fig. 6.
18
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Purification Protocol
1. Equilibrate the cartridge with 10 column volumes
(CV) of bind/wash buffer at 3 ml/min (10 ml/min).
2. Load the sample lysate at 1 ml/min (5 ml/min).
3. Wash the cartridge with 10 CV of bind/wash
buffer at 3 ml/min (10 ml/min).
4. Equilibrate cartridge with 2 CV of elution buffer
at 3 ml/min (10 ml/min)*.
5. Stop flow and incubate for 30 minutes**.
6. Elute purified protein with 5 CV of elution buffer
at 3 ml/min (10 ml/min).
* Alternative elution protocol: Pump 5 CV of elution buffer at
0.1 ml/min (0.5 ml/min) at room temperature.
**Increased incubation time, 2–4 hr at 25°C or 12–24 hr at 4°C,
may be necessary for complete cleavage of some proteins.
21
5. Remove the supernatant, and filter through a
0.45 µM filter before applying to the cartridge.
Section 8
Cartridge Preparation and
Purification Protocol
To prepare a cartridge, remove the end plugs and
connect the cartridge to the chromatography system. The
cartridge is now ready for use. The following 1 ml
cartridge purification protocol is a general guideline
for first time users. Flow rates for 5 ml cartridges are
shown in parentheses. The kinetics of cleavage are
target protein specific and may require optimization
to maximize yield. See Figure 6 for a representative
purification profile using the following protocol. Refer
to the Profinity eXact System Manual for complete
details.
20
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Section 9
Scaling Up
Bio-Scale Mini cartridges are available in 1 ml and
5 ml cartridge formats. The Profinity eXact resin is
also available in 10 ml bottles. For quick scale-up,
two or more cartridges may 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 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,
and Storage
Protein cross-contamination, frit clogging, and
increased backpressure may result from cartridge
overuse. It is recommended to dispose of a
23
Fig. 6. Native purification. Purification of Profinity eXact-tagged
Maltose Binding Protein from the soluble fraction using a BioLogic
DuoFlow system. 5 ml of lysate (5 CV) prepared from 50 ml of E. coli
culture was loaded onto a 1 ml cartridge. The cartridge was washed
with 10 CV of bind/wash buffer, and purified protein was eluted with
5 CV of elution buffer following a 30 min cleavage incubation. The
purified product was determined to be 99.5% pure by Quantity
One®software analysis. Lane 1, Precision Plus Protein unstained
standards; lane 2, soluble lysate; lane 3, flow-through; lane 4, wash;
lane 5, purified product.
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
AU
10.00 20.00 30.00 40.00
Min.Tenth
Tube #: 1 2 3 4 567 9 1112
22
kD
250
150
100
75
50
37
25
20
15
10
Lane Order
1 2 3 4 5
Cleavage
Incubation
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Note: The resin may also be cleaned with
0.1 M NaOH. Limit exposure time to 3 hours
and immediately neutralize with 5 CV of
bind/wash buffer. Regeneration of the resin
with phosphoric acid is still necessary, as
NaOH alone will not efficiently remove the
Profinity eXact tag from the resin.
25
cartridge after five purifications, however, the following
regeneration procedure may be used to prolong the
useful lifespan of a cartridge. In addition, in order to
avoid cross-contamination it is recommended that
single cartridges be designated for single proteins.
In order to reuse a cartridge, the Profinity eXact tag,
which is bound tightly to the functional ligand,
MUST be removed. Therefore, to maintain good
cartridge flow properties, and to prepare the cartridge
for a subsequent purification, it is recommended
that the cartridge be regenerated after each use.
For the 1 ml cartridges, run the regeneration protocol
at 3 ml/min. For the 5 ml cartridges, run the
regeneration protocol at 10 ml/min.
Regeneration and Storage
1. After elution of the purified protein, wash the
cartridge with 5 CV of bind/wash buffer.
2. Regenerate the cartridge with 3 CV of 100 mM
phosphoric acid.
3. Neutralize the cartridge with 5 CV of bind/wash
buffer.
24
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Problem Possible Cause Solution
Precipation during Binding capacity of Load less sample
purification cartridge exceeded
Protein aggregation Include detergent or
glycerol in buffers
27
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 0.45 µM filter prior
to application
Sample too viscous Add nuclease to lysate to
degrade DNA
No target protein Low level of target Check expression level by
in eluant protein in starting SDS-PAGE
material
Target protein not Used resin must be
binding regenerated before reuse.
Follow regeneration protocol
in Section 10
Inaccessible tag Clone into pPAL’s Spe I site
Target protein in Intrinsic cleavage Ensure no chloride ions are
flowthrough or wash present in lysate or bind/wash
fractions buffers. Perform purification
at 4°C. Clone into pPAL’s
Spe I site
Uncomplete elution No or slow cleavage Lengthen cleavage incubation
step. Clone into pPAL’s Spe I
site
26
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Section 13
References
Ruan, B et al. Engineering subtilisin into a fluoride-
triggered processing protease useful for one-step
purification. Biochemistry 43, 14539-46 (2004)
Section 14
Legal Notices
B-PER is a trademark of Pierce Biotechnology, Inc.
BugBuster is a trademark of Novagen, Inc.
CelLytic is trademark of Sigma-Aldrich
Biotechnology LP and Sigma Aldrich Co.
cOmplete is a trademark of a member of the Roche
Group.
FastBreak is trademark of Promega Corporation.
FPLC is a trademark of GE Healthcare.
ReadyPreps is a trademark of Epicentre
Technologies Corporation.
I
29
Section 12
Ordering Information
Cartridges
Catalog # Description
732-4646 Bio-Scale Mini Profinity eXact cartridge,
2 x 1 ml cartridge
732-4648 Bio-Scale Mini Profinity eXact cartridge,
1 x 5 ml cartridge
For the most up to date list of other cartridge offerings, please visit
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 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 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
28
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Profinity eXact™vectors, tags, and resins are
exclusively licensed under patent rights of Potomac
Affinity Proteins. This product is intended for
research purposes only. For commercial applications
or manufacturing using these products, commercial
licenses can be obtained by contacting the Life
Science Group Chromatography Marketing
Manager, Bio-Rad Laboratories, Inc., 6000 Alfred
Nobel Drive, Hercules, CA 94547, Tel (800) 4BIORAD.
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