129
at 529 nm; therefore, the fractions containing LH 2 -AMP can
be separated from the fractions containing LH 2 .
18. LH 2 -AMP can be stored at −80 °C for several months.
19. The adenylation activity of BD1/Donor should be fully maintained while that of BD2/Acceptor should be suppressed as
much as possible. Therefore, these kinetics are advised to be
determined before performing FlimPIA.
20. When the kinetics for one substrate are determined, the concentration of the other substrate should be maintained under
saturating conditions.
21. The oxidative luminescent activity of BD1/Donor should be
suppressed, while that of BD2/Acceptor should be fully maintained. Therefore, these LH 2 -AMP kinetics are preferably
determined before performing FlimPIA.
22. The maximum S/B ratio is usually observed 1 s after injecting
substrates. The longer reaction time gives rise to higher background luminescence due to the accumulation of LH 2 -AMP in
the reaction mixture.
23. When the substrate solution is injected into the reaction mixture, rapid mixing gives reliable result of light emission.
Therefore, we recommend using a tube-based luminometer
such as NU-2600 with stirring the mixture at top speed.
However, a 96-well luminometer also gives a reasonably good
result.
24. Dehydroluciferyl-AMP (L-AMP), which is spontaneously produced from LH 2 -AMP, acts as a competitor of LH 2 -AMP and
inhibits light emission. Coenzyme A converts L-AMP to dehydroluciferyl-coenzyme A, which does not act as a competitor
of LH 2 -AMP.
25. We previously used higher concentration (20 mM) of
ATP. The ATP-based K m of the Acceptor is increased due to
K529Q mutation, while that of the Donor should remain
unchanged. In the optimized FlimPIA, the final concentration
of ATP is reduced to 1 mM, so that the residual adenylation
activity of the Acceptor is selectively suppressed to lower the
background signal.
Acknowledgment
We thank B. Branchini for providing inspiration for the project.
This project was supported by SENTAN, JST, Japan, and partly by
Kikkoman Co., Japan, and the “Leave a Nest” Microtech Nichion
award.
Sensitive Protein-Protein Interaction Assay
at 529 nm; therefore, the fractions containing LH 2 -AMP can
be separated from the fractions containing LH 2 .
18. LH 2 -AMP can be stored at −80 °C for several months.
19. The adenylation activity of BD1/Donor should be fully maintained while that of BD2/Acceptor should be suppressed as
much as possible. Therefore, these kinetics are advised to be
determined before performing FlimPIA.
20. When the kinetics for one substrate are determined, the concentration of the other substrate should be maintained under
saturating conditions.
21. The oxidative luminescent activity of BD1/Donor should be
suppressed, while that of BD2/Acceptor should be fully maintained. Therefore, these LH 2 -AMP kinetics are preferably
determined before performing FlimPIA.
22. The maximum S/B ratio is usually observed 1 s after injecting
substrates. The longer reaction time gives rise to higher background luminescence due to the accumulation of LH 2 -AMP in
the reaction mixture.
23. When the substrate solution is injected into the reaction mixture, rapid mixing gives reliable result of light emission.
Therefore, we recommend using a tube-based luminometer
such as NU-2600 with stirring the mixture at top speed.
However, a 96-well luminometer also gives a reasonably good
result.
24. Dehydroluciferyl-AMP (L-AMP), which is spontaneously produced from LH 2 -AMP, acts as a competitor of LH 2 -AMP and
inhibits light emission. Coenzyme A converts L-AMP to dehydroluciferyl-coenzyme A, which does not act as a competitor
of LH 2 -AMP.
25. We previously used higher concentration (20 mM) of
ATP. The ATP-based K m of the Acceptor is increased due to
K529Q mutation, while that of the Donor should remain
unchanged. In the optimized FlimPIA, the final concentration
of ATP is reduced to 1 mM, so that the residual adenylation
activity of the Acceptor is selectively suppressed to lower the
background signal.
Acknowledgment
We thank B. Branchini for providing inspiration for the project.
This project was supported by SENTAN, JST, Japan, and partly by
Kikkoman Co., Japan, and the “Leave a Nest” Microtech Nichion
award.
Sensitive Protein-Protein Interaction Assay