Bio-Rad Profinity IMAC Resins User Manual
Bio-Scale™Mini
Profinity™IMAC
Cartridges, 1 and 5 ml
Instruction Manual
Catalog #
732-4610
732-4612
732-4614
Table of Contents
Section 1...Introduction........................................1
Section 2 Product Information............................2
Section 3 Connection to Low-Pressure
Chromatography Systems..................6
Section 4 Connection to Medium and High-
Pressure Chromatography Systems...8
Section 5...Buffers and Methods ..........................9
Section 6 Quick Solubility Screening
Protocols..........................................11
Section 7 Preparation of E. coli Lysates ...........15
Section 8 Preparing a Cartridge, and
Subsequent Purification....................17
Section 9 Scaling Up........................................21
Section 10 Regenerating, Cleaning,
Sanitizing, and Storage.....................22
Section 11 Troubleshooting Guide......................25
Section 12 Ordering Information.........................27
Section 13 References.......................................28
Section 14 Legal Notices ...................................29
Section 1
Introduction
Bio-Scale Mini IMAC cartridges are convenient,
disposable, prepacked low-pressure chromatographic
cartridges. Bio-Scale Mini cartridges offer both
increased run-to-run uniformity and high purity of
protein through a patent pending column design
and novel resin technology. Compatible with aqueous
buffers most commonly used for protein purification
the Bio-Scale Mini cartridges offer improved
performance for your protein separation needs.
Immobilized metal affinity chromatography (IMAC) is an
excellent chromatography technique for purification of
His-tagged proteins. The principle of IMAC is based
on the affinity histidine has for metal ions. Side chains
on the iminodiacetic acid (IDA) functional ligand
selectively bind recombinant His-tagged proteins when
the resin is charged with Ni
2+
or other metals. The
advantage of this technique is that proteins can often
be purified close to homogeneity in a single step.
Bio-Scale Mini IMAC cartridges are packed with
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Bio-Rad’s innovative Profinity™ IMAC resin.
Structural characteristics such as the polymeric
nature, optimized ligand density, and open pore
structure of the Profinity IMAC bead result in superb
mechanical strength with high stringency, low
nonspecific binding, and the ability to perform separations at high flow rates
.
Section 2
Product Information
The Bio-Scale Mini cartridges are disposable,
easy-to-use, prepacked chromatographic cartridges
supplied ready for use in convenient 1 ml and 5 ml
sizes. Cartridges are available for a variety of
chromatographic techniques, including desalting ion
exchange (IEX), affinity (AC), mixed-mode, and
hydrophobic interaction chromatography (HIC). See
Ordering Information, Section 12, for a listing of the
complete Bio-Scale Mini cartridge product line.
The Bio-Scale Mini cartridges are quickly connected
to liquid chromatography systems or luer syringes.
2
The 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.
Table 1 Bio-Scale Mini IMAC 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
Recommended flow rates 1 ml: 1–2 ml/min (240 cm/hr–480 cm/hr)
5 ml:5–10 ml/min (240 cm/hr–480 cm/hr)
Maximum flow rate 1 ml: 6 ml/min (1440 cm/hr)
5 ml: 20 ml/min (963 cm/hr)
Fittings Female luer-lock inlet and
male luer-lock 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. Profinity IMAC Resin Specifications
Functional ligand IDA
Base bead UNOsphere
Particle size range 45–90 µm
Mean particle size 60 µm
Metal ion capacity 12–30 µmol Cu
2+
/ml
Dynamic binding capacity* ≥15 mg/ml
Recommended linear flow rate 480 cm/hr
Maximum operating pressure 45 psi
Chemical compatibility See table
Storage 4°C to ambient temperature
Shelf life in 20% EtOH >1 year at ambient temperature
Operational temperature 4–40°C
* Q10% determination of 1.8 mg/ml (His)6-tagged pure protein (32 kD).
Note: Dynamic binding capacity will vary from protein to protein.
Profinity IMAC cartridges are compatible with aqueous
buffers most commonly used with IMAC purification
techniques. For a complete list of chemical
compatibilities, refer to the Profinity IMAC
instruction manual, online at http://www
.biorad.com/cmc_upload/Literature/164111/10001677
B.PDF.
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Table 3. Buffer and Chemical Compatibilities for
Profinity IMAC cartridges
Reagent Stability
Buffer Reagents
Tris 50 mM
HEPES 50 mM
MOPS 50 mM
Sodium or potassium phosphate 50 mM
Chelating Agents
EDTA, EGTA 0.1 mM
Sulfhydryl Reagents
β−Mercaptoethanol 30 mM
DTT 5 mM
TCEP 10 mM
Detergents
Nonionic detergents (Triton, Tween, NP-40) 5%
Cationic detergents (CTAB) 1% (care must be taken to
avoid protein precipitation)
Zwitterionic detergents (CHAPS, CHAPSO) 5%
Anionic detergents (SDS, Sarkosyl) 1%
Denaturing Agents
Guanidine-HCl 6 M
Urea 8 M
Other Additives
NaCl 2 M (at least 300 mM NaCl
should be included in buffers)
MgCl
2
100 mM (HEPES or Tris
should be used to prevent
precipitation)
CaCl
2
10 mM (HEPES or Tris
should be used to prevent
precipitation)
Glycerol 20%
Ethanol 20%
Imidazole 25 mM in wash buffer
500 mM for elution
Citrate 80 mM
* Profinity IMAC binding capacities are unaffected with typical reagents used for
His-tagged protein purification, up to the concentrations given.
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Section 3
Connection to
Low-Pressure
Chromatography Systems
Bio-Scale Mini cartridges are ideal for use with any
low-pressure chromatography system, including
Bio-Rad’s BioLogic LP system, Econo gradient
pump, and Model EP-1 Econo pump. For optimum
performance, we recommend choosing biocompatible
low-pressure tubing with an inner diameter (ID) of
1.6 mm.
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Fig. 1. Luer fittings and column: a cartridge should be
mounted vertically with the arrow on the cartridge pointing
downward.
To order compatible polypropylene 1.6 mm barb to
male and female luer end fittings, refer to the
ordering information located in Section 12 of this
manual.
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