artifacts, the deviation between the experimental windows of
the direct and competitive measurements should not
exceed 20%.
8. During dialysis samples are diluted to some extent. Never use
DTT as a reducing agent during ITC measurement. The
amount of samples needed for the measurements depends on
the type of instrument.
9. Be aware of avoiding air bubbles in the sample cell and in the
syringe. Note that larger volumes of samples are needed to fill
the sample cell and the syringe than the actual volumes of the
cell and syringe.
10. If the change of heat is small, measure the interaction of
interest on a different temperature, as ΔH is temperaturedependent.
11. You should use only the inner 60 cells (minimizing the potential for thermal gradient and also for evaporation at the edges).
At least 4–5 parallel measurements is recommended for individual interactions.
12. Pipet FuGene HD reagent directly to the solution, not to the
plastic wall of the tube.
13. Regarding the small cell number (20,000/well), try to avoid to
disturbing the cell monolayer. Remove the medium slowly
with extra care to maintain it.
14. For higher expression level of the protein(s) of interest, incubate the cells for 36–42 h before the measurement.
15. Always prepare Nano-Glo Live Cell reagent freshly. The furimazine component will decay to some extent. Do not freezethaw the Nano-Glo Live Cell Substrate more than 4–5 times.
16. The luminescence signal first increases, reaches a plateau, then
begins to decrease. This usually takes 15–30 min, depending
on the number of freeze–thaw cycles.
17. Always add water prior to EGF (or any other compound). Be as
fast as possible!
Acknowledgments
We thank Dr. Gergo ˝ Go ´ gl for reading the manuscript. We also
thank Vikto ´ ria Bilics for contributing in the FP measurement.
This work was supported by the National Research, Development
and Innovation Office (NKFIH) grants K119359 (to LN). MAS
was supported through the New National Excellence Program of
the Hungarian Ministry of Human Capacities. Project no. 20181.2.1-NKP-2018-00005 has been implemented with the support
provided from the National Research, Development and
190
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray
the direct and competitive measurements should not
exceed 20%.
8. During dialysis samples are diluted to some extent. Never use
DTT as a reducing agent during ITC measurement. The
amount of samples needed for the measurements depends on
the type of instrument.
9. Be aware of avoiding air bubbles in the sample cell and in the
syringe. Note that larger volumes of samples are needed to fill
the sample cell and the syringe than the actual volumes of the
cell and syringe.
10. If the change of heat is small, measure the interaction of
interest on a different temperature, as ΔH is temperaturedependent.
11. You should use only the inner 60 cells (minimizing the potential for thermal gradient and also for evaporation at the edges).
At least 4–5 parallel measurements is recommended for individual interactions.
12. Pipet FuGene HD reagent directly to the solution, not to the
plastic wall of the tube.
13. Regarding the small cell number (20,000/well), try to avoid to
disturbing the cell monolayer. Remove the medium slowly
with extra care to maintain it.
14. For higher expression level of the protein(s) of interest, incubate the cells for 36–42 h before the measurement.
15. Always prepare Nano-Glo Live Cell reagent freshly. The furimazine component will decay to some extent. Do not freezethaw the Nano-Glo Live Cell Substrate more than 4–5 times.
16. The luminescence signal first increases, reaches a plateau, then
begins to decrease. This usually takes 15–30 min, depending
on the number of freeze–thaw cycles.
17. Always add water prior to EGF (or any other compound). Be as
fast as possible!
Acknowledgments
We thank Dr. Gergo ˝ Go ´ gl for reading the manuscript. We also
thank Vikto ´ ria Bilics for contributing in the FP measurement.
This work was supported by the National Research, Development
and Innovation Office (NKFIH) grants K119359 (to LN). MAS
was supported through the New National Excellence Program of
the Hungarian Ministry of Human Capacities. Project no. 20181.2.1-NKP-2018-00005 has been implemented with the support
provided from the National Research, Development and
190
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray