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Choosing which interactant to immobilize

Many factors can influence the decision on which interactant to attach to the surface and which attachment approach to use. Some or all of the following considerations may be relevant in any individual situation:

• With covalent immobilization methods, ligand is attached once for the whole assay, so ligand consumption is kept to a minimum. On the other hand, assay development work will probably be necessary to establish suitable conditions for both immobilization and ligand regeneration, costing time and material.
• With captured ligand, using capturing kits or well-characterized reagents, conditions for immobilization and regeneration of the capturing molecule are usually known so assay development requirements are reduced. On the other hand, fresh ligand needs to be captured for each analysis cycle, taking more time for the assay and requiring larger amounts of ligand.
• Tagged recombinant proteins can often be captured using interaction with the tag. Kits and sensor chips are available from Biacore for capture with a number of common tags.
• If you want to hold one interactant constant and vary the other, it is usually most convenient to attach the constant interactant to the surface. A capturing approach is valuable if the constant interactant cannot be attached to the surface or if you want to vary both interactants, since the captured ligand can be changed between cycles.
• Large molecules such as proteins are often easier to immobilize than small molecules, and also run less risk of the attachment interfering with the interaction being studied. On the other hand, small molecules give inherently lower sample responses that may be more difficult to measure with confidence.
• Capture is often unsuitable for attaching small molecules since the approach requires two separate binding sites on the molecule, one for the capturing interaction and one for the interaction being studied.
• Viruses and cells should be injected as sample, not immobilized on the surface. The detection decreases exponentially with the distance from the sensor surface and an interaction on the surface of an immobilized virus or cell may be too distant from the surface to give a detectable response.

See also

List of help files