On a Small Scale

Step 1 - To the Ligand-attached Magnetic Agarose Beads (bottom of tube; black), a solution containing the Ligand Target (T) substance is added.

Step 2 - The Magnetic L-Agarose Beads are mixed with the T-containing solution.

Step 3 - The Magnetic L-Agarose Beads are quickly localized with a magnet - allowing all unbound sample to be easily decanted.

Step 4 : Add a suitable wash solution to remove any substances not specifically bound to the ligand (L).

Step 5 : Allow the wash solution to penetrate and wash the suface of the beads for a few minutes.

Step 6 : Quickly localize the beads on the side of the tube using a suitable magnet & decant the supernatant.

Step 7: Add a solution which will break the T-L bond so that the Target Substance is released into the solution from the Magnetic Agarose Bead.

Step 8: Allow the T-L bond-breaking solution to contact all bead surfaces for an appropriate period of time ( a function of T-L binding constant, residence time and some other factors).

Step 9: Quickly localize the beads on the side of the tube using a suitable magnet & decant and SAVE the T-containing SUPERNATANT .

Step 10 : You've completed your isolation & purification of substance T from a complex mixture- much FASTER than you could have with the same ligand on a standard drip-drip-drip affinity column - often with much better yields !

Larger Scale Tips

Photo #1

a) Assemble the necessary magnet (s), container (s) and protective film for the tip of the magnet. Illustrated in the photo are : a plastic 30 ml test tube, a suitable "5lb. pull or lift" magnet with telescoping extension, a stir bar (having too weak a magnet) with a neodymium (NdFeB) 5 lb. disc shaped magnet for the end of the stir bar, a white plastic pot and importantly, a sheet of Saran Wrap (or equivalent).

Photo #2

b) The Saran Wrap( sterilized before use) is used to cover the tip of the magnet. After folding the ends of the Saran Wrap back up the handle of the magnet retrieval device and secured with a twist tie ( also sterilized before use; copper or SS wire is one option)

Photo #3

a)Once the magnet has been wrapped with an easily removable film, the magnet can now be swept through a large volume of liquid containing both Magnetic L-Agarose Beads and the T-containing complex mixture. ( Note: If a removable film is not used to cover the magnet, then it will be extremely difficult to remove the magnetic agarose beads once they attach to the magnet ! )

b) This photo shows how the Saran film-covered magnet can be swept through a test tube and the magnetic L-agarose beads collected (with bound T). This is ann altrnative to holding the magnet outside the tube - either on the side or the bottom.

Photo #4

Alternatively, a larger than test-tube-sized volume of T-containing mixture and Magnetic L-Agarose Beads can be swept with the magnet and all the beads collected on the end of the magnet.

Photo #5

a)After collecting all of the L-T Magnetic Agarose Beads (black) on the tip of the magnet, the protective Saran Film can be released once the magnet has been placed in a convenient collection vessel (white pot for illustration purposes).

b) A stream of distilled water or buffer can then be aimed at the beads on the unwrapped Saran film. The beads will readily wash off the film and are now ready for the next step in the process.

Note: It is highly recommended that the magnetic beads be redispersed (i.e. not kept bound to the magnet ) during the WASH and the L-T BOND- BREAKING steps. Otherwise, good washing and L-T bond breaking ( & T recovery) may be compromised if the beads are kept tightly bound to the magnet during those steps.

“How To” Protocol for Magnetic Bio-Affinity Separations:

a successful magnetic bioseparation are discussed below followed by a step-by-step protocol. The low cost, speed and simplicity of this protocol compared to a comparable affinity column technique is dramatic.

A. The Magnetic Bead:

  1. Shape: It is important that the bead be spherical. Irregular shaped beads will tend to clump during recovery and redisperse poorly during subsequent steps.

  2. No Non-Specific Bonding: If the composition of the bead or method of ligand attachment produces ionic or hydrophobic groups - these will result in the binding of substances other than those having an affinity for the ligand. The highly porous agarose gel found in BioScience Magnetic Beads, is an ideal neutral polysaccharide having no ionic groups or hydrophobic groups and also readily allows for convenient ligand immobilization .

  3. Ligand Binding Capacity vs. magnetic composition: The bead must have sufficient magnetite to be attracted by a suitable magnet but excess magnetite will reduce the potential binding capacity per unit volume of beads. At the other extreme, if some beads in a population do not have sufficient magnetite, they will tend to be lost during wash steps.

  4. Buoyancy: The magnetic beads should have good suspension characteristics in water so they can be conveniently stirred with just a stir bar or overhead stirrer but do not require expensive special equipment.

B) The Magnet:

  1. Type: A permanent magnet is highly preferred to avoid loss of magnetism over time or the need to remagnetize the magnet.

  2. Strength: A magnet having a 5 lb. lift capacity is optimal for recovering Magnetic BioScience Agarose beads. The strength of the magnetic field produced by any magnet decreases as the inverse square of the distance from the magnet.

  3. Composition: Neodymium magnets are preferable to most alternatives. These magnets are made from a combination of metals and rare earth minerals which make them both strong and compact. This type of magnet is supplied free with qualifying orders of Magnetic BioScience Beads.

  4. Shape: The magnet shape can be adjusted to the method and scale of use. Rod, disk, plate or doughnut shapes are all common.

  5. Size: As with shape, above, the size of the magnet can be scaled to the recovery method used.

  6. Safety: Magnets can damage magnetic media like floppy disks, credit cards, magnetic I.D. cards, cassette tapes, video tapes, and similar products. They can also damage televisions, VCR’s computer monitors and other CRT electronics. As a result, a magnet should never be placed near any of these products. A Neodymium Magnet should never be brought near a person with a pacemaker or similar medical device because it could alter it’s operation. Neodymium Magnets should not be either machined or burned and will lose their magnetic properties if heated above 80°C.