Streptavidin-coated donor beads

The first consideration when setting up an AlphaScreen assay is the choice of an assay format In most cases, the decision will depend on the interaction partners under investigation and on the biological tools available for their detection. The interaction partners can be coupled to the beads directly via reductive amination of reactive aldehyde groups, similar to the immobilization on a Biacore sensor chip (see above). The usefulness of this approach is limited by the reaction conditions, which may not be appropriate for maintaining the biologically active conformation of the biomolecule. Therefore the biomolecule of interest is usually not coupled to the beads directly, but instead captured via an antibody, also preventing steric hindrance. While not strictly necessary, it is often convenient to use a biotinylated molecule which can be captured by streptavidin-coated donor beads. 

The components involved are streptavidin-coated donor beads and anti-GST acceptor beads (Perkin Elmer LAS, Boston, USA) and - as the biological system -the PKA subunits as cAMP sensors. Upon binding of cAMP to each regulatory (R) subunit, the PKA holoenzyme dissociates releasing the catalytic (C) subunits. 

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