advisable to remember that the determination of the dissociation rate from such limited dissociation is based primarily on
the assumption that the response following complete dissociation will be identical to that recorded prior to the association
step (i.e., the response in step (c) in Fig. 1). Any instrument
drift or residual nonspecific binding can therefore have a significant effect on the dissociation rate constant determined.
9. Regeneration and subsequent reuse of biosensors offers the
user considerable cost savings. The immobilized molecule
must be stable under the regeneration conditions employed
(generally high or low pH, high salt concentration or added
detergent) and must retain binding capacity over several regeneration cycles. In addition, bound molecules must of course be
completely removed by the regeneration process.
10. In rare cases, the amount of nonspecific binding observed with
a loaded biosensor can be greater than that seen on an
unloaded one. The presence of a slow phase in the association
step that never reaches equilibrium followed by incomplete
dissociation is an indication that there are problems with nonspecific binding of the protein to the sensor surface.
11. SSA biosensors are super streptavidin sensors. They have
higher density of streptavidin on the surface compared to the
SA sensors. This allows for a higher binding signal, and interactions over a larger surface area of the biosensor will be
specific, thus reducing nonspecific binding. Note however
that SSA biosensors are much more expensive than SA.
12. This approach is particularly useful if one of the proteins gives
such a poor response that it is not possible to obtain a dissociation constant using the standard protocol described in Subheading 3.1.
13. The coupling is defined as weak because in the case of perfect
coupling, the affinity of the KH3KH4 construct would be
equal to the product of the affinities of the RNA:KH3KH4
(DD) and RNA:KH3(DD)KH4 complexes (~1.5 μM
and ~0.9 μM), i.e., ~1.4 pM.
14. The alternative binding pathway, i.e., binding of a second
protein to the same RNA, would require a significantly higher
affinity for the two interactions because the concentrations of
protein and RNA used in our experiments are low compared
with the K d s for the binding of the individual domains.
15. These equations only apply if the conformational change is very
much faster than the bimolecular step over the range of protein
concentrations being examined. If the bimolecular step is faster
than the conformational change under all conditions, then
there would be two kinetic phases with the fast phase varying
linearly with protein concentration and the slow process
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Stephen R. Martin et al.
the assumption that the response following complete dissociation will be identical to that recorded prior to the association
step (i.e., the response in step (c) in Fig. 1). Any instrument
drift or residual nonspecific binding can therefore have a significant effect on the dissociation rate constant determined.
9. Regeneration and subsequent reuse of biosensors offers the
user considerable cost savings. The immobilized molecule
must be stable under the regeneration conditions employed
(generally high or low pH, high salt concentration or added
detergent) and must retain binding capacity over several regeneration cycles. In addition, bound molecules must of course be
completely removed by the regeneration process.
10. In rare cases, the amount of nonspecific binding observed with
a loaded biosensor can be greater than that seen on an
unloaded one. The presence of a slow phase in the association
step that never reaches equilibrium followed by incomplete
dissociation is an indication that there are problems with nonspecific binding of the protein to the sensor surface.
11. SSA biosensors are super streptavidin sensors. They have
higher density of streptavidin on the surface compared to the
SA sensors. This allows for a higher binding signal, and interactions over a larger surface area of the biosensor will be
specific, thus reducing nonspecific binding. Note however
that SSA biosensors are much more expensive than SA.
12. This approach is particularly useful if one of the proteins gives
such a poor response that it is not possible to obtain a dissociation constant using the standard protocol described in Subheading 3.1.
13. The coupling is defined as weak because in the case of perfect
coupling, the affinity of the KH3KH4 construct would be
equal to the product of the affinities of the RNA:KH3KH4
(DD) and RNA:KH3(DD)KH4 complexes (~1.5 μM
and ~0.9 μM), i.e., ~1.4 pM.
14. The alternative binding pathway, i.e., binding of a second
protein to the same RNA, would require a significantly higher
affinity for the two interactions because the concentrations of
protein and RNA used in our experiments are low compared
with the K d s for the binding of the individual domains.
15. These equations only apply if the conformational change is very
much faster than the bimolecular step over the range of protein
concentrations being examined. If the bimolecular step is faster
than the conformational change under all conditions, then
there would be two kinetic phases with the fast phase varying
linearly with protein concentration and the slow process
366
Stephen R. Martin et al.