233
using FKBP1A-NTEV or FKBP1A-CTEV as negative controls
as FKBP1A represents a small protein with a defined number
of interactions described [44]. FKBP1A was used in split TEV
assays [40], and also by other split protein approaches. Further,
we have successfully used the GCN4- coiled coil domain
(GCN4cc) as interaction control [9, 41]. Another strategy
may be applying a truncated form of an interacting partner
that lacks the defined interaction domain(s). However, it is
crucial to validate its correct use first.
6. We suggest to store DNA plasmid aliquots at −20 °C. Avoid
unnecessary freeze-thaw cycles as this decreases DNA concentration and quality.
7. Seeding the cells can be done on the previous day or in the
morning of the day the experiment is planned. When seeding
on the same day, increase the amount of cells by roughly 50%
and wait at least 3–4 h to let the cells attach to the surface of
the plate.
8. It is also possible to prepare the DNA mix on the previous day.
If done so, freeze the DNA as small volumes may evaporate at
4 °C.
9. To optimize transfection conditions, start with adapting plasmid amounts, i.e., increase/decrease DNA amounts, and/or
change ratios of the candidate X vs. candidate Y vs. reporter
plasmids. Further, we find changing cell densities and/or
Lipofectamine 2000 concentrations helpful.
10. Agonists can be added in a 10× or 20× concentration diluted
in assay medium. Note that both agonists and antagonists are
frequently diluted in DMSO, which must not exceed 1% of the
total volume.
11. For RTKs, we regularly incubate for 20 h to obtain stable
inducible readings. For GPCRs, we noted, however, that
shorter incubation periods of 6–8 h yield better readings. To
determine the optimal time window for a given inducible PPI,
we also suggest to run an online luciferase split TEV assay
before investing into a dose response assay.
12. When opting for a fluorescent reporter, such as UAS_EGFP,
do not lyse the cells, and proceed with acquiring the fluorescent signal using a fluorescence reader or an automated
microscope.
13. When acquiring luciferase signal intensities using the Mithras
device from Berthold Technologies, we apply a time integration protocol, which measures each well for 2 s. Substrates are
added by automated injection across the entire plate, followed
by an orbital shake for 10 s and an incubation time of 10 min,
followed by reading the signals. This process is performed first
Characterising Dynamic PPIs Using Split TEV
using FKBP1A-NTEV or FKBP1A-CTEV as negative controls
as FKBP1A represents a small protein with a defined number
of interactions described [44]. FKBP1A was used in split TEV
assays [40], and also by other split protein approaches. Further,
we have successfully used the GCN4- coiled coil domain
(GCN4cc) as interaction control [9, 41]. Another strategy
may be applying a truncated form of an interacting partner
that lacks the defined interaction domain(s). However, it is
crucial to validate its correct use first.
6. We suggest to store DNA plasmid aliquots at −20 °C. Avoid
unnecessary freeze-thaw cycles as this decreases DNA concentration and quality.
7. Seeding the cells can be done on the previous day or in the
morning of the day the experiment is planned. When seeding
on the same day, increase the amount of cells by roughly 50%
and wait at least 3–4 h to let the cells attach to the surface of
the plate.
8. It is also possible to prepare the DNA mix on the previous day.
If done so, freeze the DNA as small volumes may evaporate at
4 °C.
9. To optimize transfection conditions, start with adapting plasmid amounts, i.e., increase/decrease DNA amounts, and/or
change ratios of the candidate X vs. candidate Y vs. reporter
plasmids. Further, we find changing cell densities and/or
Lipofectamine 2000 concentrations helpful.
10. Agonists can be added in a 10× or 20× concentration diluted
in assay medium. Note that both agonists and antagonists are
frequently diluted in DMSO, which must not exceed 1% of the
total volume.
11. For RTKs, we regularly incubate for 20 h to obtain stable
inducible readings. For GPCRs, we noted, however, that
shorter incubation periods of 6–8 h yield better readings. To
determine the optimal time window for a given inducible PPI,
we also suggest to run an online luciferase split TEV assay
before investing into a dose response assay.
12. When opting for a fluorescent reporter, such as UAS_EGFP,
do not lyse the cells, and proceed with acquiring the fluorescent signal using a fluorescence reader or an automated
microscope.
13. When acquiring luciferase signal intensities using the Mithras
device from Berthold Technologies, we apply a time integration protocol, which measures each well for 2 s. Substrates are
added by automated injection across the entire plate, followed
by an orbital shake for 10 s and an incubation time of 10 min,
followed by reading the signals. This process is performed first
Characterising Dynamic PPIs Using Split TEV