231
Lysed cells can be stored at −20 °C for up to 4 weeks without
losing significant levels of signal intensity (see Note 12).
18. Perform the Dual luciferase readout. Add 30 μl of Firefly substrate into each well (from commercial Dual Luciferase Kit
prepared as indicated by the manufacturer), read the plate
using a luciferase reader. Add 30 μl of Renilla substrate into
each well, read the plate (see Note 13). Alternatively, self- made
substrates may be used (see Note 14).
19. Calculate mean, standard deviations and standard errors of the
mean using an Excel spreadsheet. Generate a bar graph to
visualize the data. For dose-response analyses, we recommend using the dose response curve package ‘drc’ in R (see
Note 15). Alternatively, the GraphPad Prism program can be
used, which allows standard visualizations of dose response
curves.
20. Advice for troubleshooting addressing critical steps can be
found in Table 1.
Table 1
Troubleshooting advice
Step
Problem
Solution
4, 10
Low signals in general (both
firefly and Renilla)
Do not use antibiotics as they might interfere with the
transfection reagent
12, 16
Good transfection efficiency,
good Renilla signals, but
low firefly signals:
• ORF-NTEV
• ORF-NTEV-tevS-GV
• ORF-CTEV fusion
protein may not be
expressed properly
Although the optical transfection control is fine as
indicated by for example EGFP expression, the candidate
fusion in question may be poorly expressed
• Verify protein expression using Western blotting
• Try to swap candidate proteins for NTEV and CTEV
fragments. If using the cytoplasmic assay, you may also
place the NTEV moiety to either end. Protein
expression, however, also depends on the target protein
chosen
16
Low firefly signals for
inducible PPIs
Stimulation time may vary for a given receptor or any other
inducible PPI. Therefore, we suggest to run an online
luciferase split TEV assay to obtain the best activation
window. If no appropriate reader is available, monitor
assay activity from 6 to 20 h in an hourly or two-hourly
mode after the agonist has been added
18
Low luminescence signals,
in particular for firefly
luciferase
Double-check the functionality of the luciferase buffers.
This is of particular importance for the firefly luciferase
buffer containing ATP, which can decay quickly. To do
so, we prepare test lysates from cells that constitutively
express both firefly and Renilla luciferases. These lysates
can easily be stored at −80 °C for several months
Characterising Dynamic PPIs Using Split TEV
Lysed cells can be stored at −20 °C for up to 4 weeks without
losing significant levels of signal intensity (see Note 12).
18. Perform the Dual luciferase readout. Add 30 μl of Firefly substrate into each well (from commercial Dual Luciferase Kit
prepared as indicated by the manufacturer), read the plate
using a luciferase reader. Add 30 μl of Renilla substrate into
each well, read the plate (see Note 13). Alternatively, self- made
substrates may be used (see Note 14).
19. Calculate mean, standard deviations and standard errors of the
mean using an Excel spreadsheet. Generate a bar graph to
visualize the data. For dose-response analyses, we recommend using the dose response curve package ‘drc’ in R (see
Note 15). Alternatively, the GraphPad Prism program can be
used, which allows standard visualizations of dose response
curves.
20. Advice for troubleshooting addressing critical steps can be
found in Table 1.
Table 1
Troubleshooting advice
Step
Problem
Solution
4, 10
Low signals in general (both
firefly and Renilla)
Do not use antibiotics as they might interfere with the
transfection reagent
12, 16
Good transfection efficiency,
good Renilla signals, but
low firefly signals:
• ORF-NTEV
• ORF-NTEV-tevS-GV
• ORF-CTEV fusion
protein may not be
expressed properly
Although the optical transfection control is fine as
indicated by for example EGFP expression, the candidate
fusion in question may be poorly expressed
• Verify protein expression using Western blotting
• Try to swap candidate proteins for NTEV and CTEV
fragments. If using the cytoplasmic assay, you may also
place the NTEV moiety to either end. Protein
expression, however, also depends on the target protein
chosen
16
Low firefly signals for
inducible PPIs
Stimulation time may vary for a given receptor or any other
inducible PPI. Therefore, we suggest to run an online
luciferase split TEV assay to obtain the best activation
window. If no appropriate reader is available, monitor
assay activity from 6 to 20 h in an hourly or two-hourly
mode after the agonist has been added
18
Low luminescence signals,
in particular for firefly
luciferase
Double-check the functionality of the luciferase buffers.
This is of particular importance for the firefly luciferase
buffer containing ATP, which can decay quickly. To do
so, we prepare test lysates from cells that constitutively
express both firefly and Renilla luciferases. These lysates
can easily be stored at −80 °C for several months
Characterising Dynamic PPIs Using Split TEV