proceed for another hour at 37
C. Alkaline phosphate and
restriction enzymes are removed in the following gel
extraction step.
13. When using ampicillin selection, the 1 h incubation step at
37
C can be omitted and the cells directly spread on selection
plates.
14. These numbers are indicative and must be adapted to each cell
line, including HeLa cells from different lab origin.
15. As a negative control, we recommend pairing each the two
tested proteins with an unrelated protein. Whereas using
NBirA*-GFP as an unrelated protein worked fine for us, we
recommend not using CBirA*-GFP as an additional control
protein as it always yielded considerable background biotinylation in our hands. This is likely due to the very high expression
level of CBirA*-GFP that probably leads to spontaneous
re-association of CBirA* with NBirA* [8]. Other unrelated
CBirA* fusions worked fine for us. A powerful negative control
for MS analysis is a split-BioID or BioID experiment (or a pool
of experiments) applied to two interacting proteins that are
unrelated to the proteins of interest.
16. Doxycycline is necessary if using tet-responsive element–regulated plasmids in tet-on cell lines.
17. Samples may be stored at À20
C at that point.
18. If using fluorescence-based detection and Tween-20 as a
source of autofluorescence, a fourth short wash of the membrane with plain PBS is recommended before detection.
19. We had good experience with the 9E10 (dilution 1:1000) and
M2 (dilution 1:500) mouse monoclonal antibodies directed
against myc and FLAG, respectively.
20. If detection of the fusion proteins proves to be challenging,
incubation of the primary antibodies overnight at 4
C may
yield stronger signals.
21. Cell lysates can be very viscous. If needed the syringe can be
filled the first few times without the needle.
22. These parameters worked well for a Bioruptor plus sonification
device from Diagenode and are given as guidance when using
another sonication bath.
23. We have tended to observe less efficient binding to the
streptavidin-coupled beads at much higher salt concentrations.
24. These numbers are for Dynabeads MyOne Streptavidin C1
beads and may need to be adjusted when using other types of
beads.
25. We chose to elute biotinylated proteins from the beads rather
than performing on-bead trypsin digestion. The latter is the
316
Cinthia Amaya Ramirez et al.
C. Alkaline phosphate and
restriction enzymes are removed in the following gel
extraction step.
13. When using ampicillin selection, the 1 h incubation step at
37
C can be omitted and the cells directly spread on selection
plates.
14. These numbers are indicative and must be adapted to each cell
line, including HeLa cells from different lab origin.
15. As a negative control, we recommend pairing each the two
tested proteins with an unrelated protein. Whereas using
NBirA*-GFP as an unrelated protein worked fine for us, we
recommend not using CBirA*-GFP as an additional control
protein as it always yielded considerable background biotinylation in our hands. This is likely due to the very high expression
level of CBirA*-GFP that probably leads to spontaneous
re-association of CBirA* with NBirA* [8]. Other unrelated
CBirA* fusions worked fine for us. A powerful negative control
for MS analysis is a split-BioID or BioID experiment (or a pool
of experiments) applied to two interacting proteins that are
unrelated to the proteins of interest.
16. Doxycycline is necessary if using tet-responsive element–regulated plasmids in tet-on cell lines.
17. Samples may be stored at À20
C at that point.
18. If using fluorescence-based detection and Tween-20 as a
source of autofluorescence, a fourth short wash of the membrane with plain PBS is recommended before detection.
19. We had good experience with the 9E10 (dilution 1:1000) and
M2 (dilution 1:500) mouse monoclonal antibodies directed
against myc and FLAG, respectively.
20. If detection of the fusion proteins proves to be challenging,
incubation of the primary antibodies overnight at 4
C may
yield stronger signals.
21. Cell lysates can be very viscous. If needed the syringe can be
filled the first few times without the needle.
22. These parameters worked well for a Bioruptor plus sonification
device from Diagenode and are given as guidance when using
another sonication bath.
23. We have tended to observe less efficient binding to the
streptavidin-coupled beads at much higher salt concentrations.
24. These numbers are for Dynabeads MyOne Streptavidin C1
beads and may need to be adjusted when using other types of
beads.
25. We chose to elute biotinylated proteins from the beads rather
than performing on-bead trypsin digestion. The latter is the
316
Cinthia Amaya Ramirez et al.