Bio-Rad Profinity eXact Purification and Tag Cleavage Consumables User Manual
Bio-Scale™Mini
Profinity eXact
™
Cartridges, 1 and 5 ml
Instruction Manual
Catalog #
732-4646
732-4648
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
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 (EX
act Affinity Cleavage T
echnology)
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
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
Table 3. Buffer and chemical compatibilities for
Profinity eXact cartridges.
Reagent
Group Description Stability
Triggering Ions F-, Cl-, N
3
-
, NO
2
-
, CO
2
-
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
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 CaCl
2
£5 mM when used with
MES, MOPS, or PIPES
buffers. Do not use with
phosphate buffers.
MgCl
2
£5 mM. Do not use with
fluoride containing
buffers. Use NaN
3
as 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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