Equipment and Materials

Magnetic carriers with immobilized affinity ligand or magnetic particles prepared from a biopolymer exhibiting affinity for the target compound(s) are used to perform the isolation procedure. Magnetic separators are necessary to recover magnetic particles from the system.

Magnetic carriers and adsorbents are commercially available and can also be prepared in the laboratory. Such materials are usually available in the form of magnetic particles prepared from various synthetic polymers, biopolymers or porous glass, or magnetic particles based on inorganic magnetic materials such as surface-modified magnetite can be used. In fact, many of the particles behave like paramagnetic or superparamagnetic ones responding to an external magnetic field, but not interacting themselves in the absence of a magnetic field. This is important due to the fact that magnetic particles can be easily resus-pended and remain in suspension for a long time. The diameter of the particles is from ca. 50 nm to ca. 10 |im. Magnetic particles having a diameter larger than ca. 1 | m can be easily separated using simple magnetic separators, while separation of smaller particles (magnetic colloids with a particle size ranging between tens and hundreds of nanometers) may require the use of high-gradient magnetic separators.

Commercially available magnetic particles can be obtained from a variety of companies. In most cases polystyrene is used as a matrix, but carriers based on cellulose, agarose, silica, porous glass or silanized magnetic particles are also available. Particles with immobilized affinity ligands are available, oligo-deoxythymidine, streptavidin, antibodies, protein A and protein G being used most often. Magnetic particles with such immobilized ligands can serve as generic solid phases to which native or modified affinity ligands can be immobilized (e.g. antibodies in the case of immobilized protein A, protein G or secondary antibodies, biotinylated molecules in the case of immobilized streptavidin or adenylated molecules in the case of immobilized oligodeoxythymidine). In exceptional cases, enzyme activity may decrease as a result of usage of magnetic particles with exposed iron oxides. In this case encapsulated microspheres, having an outer layer of pure polymer, are safer. In Table 1 is given a list of companies producing and selling magnetic particles of various types.

In the laboratory, magnetite (or similar magnetic materials such as maghemite or ferrites) particles are usually surface modified by silanization. This process modifies the surface of the inorganic particles so that appropriate function groups become available, which enable easy immobilization of affinity ligands.

Biopolymers such as agarose, chitosan, K-car-rageenan and alginate can be easily prepared in a magnetic form. In the simplest way, the biopolymer solution is mixed with magnetic particles and after bulk gel formation the magnetic gel formed is broken into fine particles. Alternatively, biopolymer solution containing dispersed magnetite is dropped into a mixed hardening solution or a water-in-oil suspension

Table 1 Examples of commerically available magnetic particles suitable for magnetic affinity separations

Manufacturer/supplier

Nameof particles Diameter Polymer Endgroups/

(fim) composition/surface activation modification possibility

Immobilized ligands

Advanced Biotechnologies, Epsom, UK

Bangs Labs, Fishers, IN, USA

Cortex Biochem, San Leandro, CA, USA

CPG, Lincoln Park, NJ, USA

Dynal, Oslo, Norway

Immunotech, Marseille, France

Merck, Darmstadt, Germany

Novagen, USA PerSeptive Biosystems, Farmingham, MA, USA

Prolabo, Fontenay-Sous-Bois, France

Promega, Madison, WI, USA

ProZyme, San Leandro, CA, USA

Qiagen

Quantum Magnetics, USA

Scigen, Sittingbourne, UK

Seradyn, Indianopolis, IN, USA

Spherotech, Libertyville, IL, USA

XM200 Microsphere 3.5

Magnacil Magnetic Microspheres MagaCell

Dynabeads M-280 Dynabeads M-450 Dynabeads M-500 Iobeads

Biobeads

Magnetight BioMag

Estapor

MagneSphere Paramagnetic Particles Magnetic beads

Ni-NTA Magnetic agarose beads Magnetic particles

M 100 M 104 M 108

Magnetic agarose Sera-Mag

1-10

Polystyrene Silica

Styrene-divinyl benzene copolymer Cellulose

Porous glass Polystyrene

-COOH

Silica

-SiOH, glyceryl, -NH2, hydrazide Tosyl activated

Polystyrene Silica

Silanized iron oxides -COOH, -NH2

SPHERO magnetic 1-4.5 particles

Polystyrene

Styrene-divinyl-benzene copolymer

Agarose

Cellulose

Agarose

Polystyrene

Oligo (dT), antibodies, streptavidin, protein A

Streptavidin, protein A, antibodies Streptavidin, protein A, protein G, oligo (dT), DEAE, CM, PEI Streptavidin, avidin

Streptavidin, oligo (dT), antibodies

Antibodies, avidin

Streptavidin

Oligo (dT)

Antibodies, protein A, protein G, streptavidin, biotin

Streptavidin

Streptavidin, protein A, protein A/G, protein G

Nitrilotriacetic acid

Streptavidin, DEAE, CM, C18, protein A, silica

Streptavidin, biotin, oligo (dT)

Streptavidin, oligo (dT)

Streptavidin, biotin, antibodies

technique is used to prepare spherical particles. Basically the same procedures can be used to prepare magnetic particles from synthetic polymers such as polyacrylamide or poly(vinylalcohol).

In one of the approaches used, standard affinity chromatography material is post-magnetized by pumping the water-based ferrofluid through the column packed with the sorbent. Magnetic material accumulates within the affinity adsorbent pores thus modifying the chromatography material into magnetic form.

Some affinity ligands (usually general binding ligands) are already immobilized to commercially available carriers (see Table 1). To immobilize other ligands to both commercial and laboratory-made magnetic particles, standard procedures used in affinity chromatography can be employed. Usually functional groups available on the surface of magnetic particles such as -COOH, -OH or -NH2 are used for immobilization; in some cases, magnetic particles are already available in the activated form (e.g. tosyl activated).

Magnetic separators are necessary to separate the magnetic particles from the system. In the simplest approach, a small permanent magnet can be used, but various magnetic separators employing strong rare-earth magnets can be obtained at reasonable prices. Commercial laboratory scale batch magnetic separators are usually made from magnets embedded in disinfectant-proof material. The racks are constructed for separations in Eppendorf microtubes, standard test tubes or centrifugation cuvettes. Some have a removable magnetic plate to facilitate washing of separated magnetic particles (Figure 1). Other types of separators enable separations from the wells of microtitration plates and the flat magnetic separators are useful for separation from larger volumes of suspensions (up to ca. 500-1000 mL).

Flow-through magnetic separators are usually more expensive and more complicated, and highgradient magnetic separators (HGMS) are typical examples (Figure 2). Laboratory-scale HGMS are constructed from a column packed with fine magnetic-grade stainless-steel wool or small steel balls placed between the poles of an appropriate magnet. The suspension is pumped through the column, and magnetic particles are retained within the matrix. After removing the column from the magnetic field, the particles are retrieved by flow and usually by gentle vibration of the column.

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