or StrepTactin- [9]coupled beads, have also been used successfully by others. Some beads are provided as pre-coated with
BSA to decrease nonspecific protein binding. We recommend
not using pre-coated beads, as BSA will later be a major source
of contamination for the MS analysis.
7. We usually prepare lysis and wash buffers freshly from stock
solutions (1 M Tris–HCl, pH 7.4 or 8, 5 M NaCl, 500 mM
EDTA pH 8, 20% SDS, 20% Triton X-100, 20% NP-40, 10%
sodium deoxycholate, 5 M LiCl, 1 M DTT).
8. Low-fluorescence PVDF membranes are required for
fluorescence-based detection of IR dyes-conjugated secondary
antibody. From all the PVDF membranes we tested, only
Immobilon
® -FL PVDF membrane from Millipore gave satisfactory results. Alternatively nitrocellulose membranes typically
yield low autofluorescence. If using chemoluminescence-based
detection, any immunoblotting membrane is fine. Detection of
biotinylated proteins with IR dye- or HRP-conjugated streptavidin works equally well.
9. Ultrafast transfer protocols (7 min) allowed by the Transblot
Turbo system from Bio-Rad or equivalent devices is sufficient
for the detection of biotinylated proteins with conjugated
streptavidin. Any other transfer method is also fine and may
yield better results when the detection of the fusion proteins is
needed, especially for large-size proteins.
10. The use of precast gels and commercial reagents such as loading buffer is optional but recommended to avoid excessive
keratin contamination when running SDS-PAGE for MS
samples.
11. We have provided split-BioID plasmids (see https://www.
addgene.org/Julien_Bethune/) that allow the bidirectional
expression of the NBirA* and CBirA* fusions in various orientations and under the control of the same tet-responsive element [8]. Of important note, these plasmids should be used in
tet-on or tet-off cell lines. The two fusion proteins can be
expressed from the same plasmid or from two separate plasmids. When testing a pair of protein for the first time, we
recommend trying all possible iterations of the fusion proteins
(NBirA* and CBirA* appended at the N- or C- terminus of
each protein of interest, and protein 1 appended to NBirA* or
CBirA*). We did observe that certain combination work significantly better than others.
12. If later many of the resulting isolated plasmids prove to be the
wrong constructs, a dephosphorylation can be included. Typically we digest the plasmids as indicated, and after 1 h, add
alkaline phosphatase buffer and enzyme and let the reaction
Context-Specific Proximity-Dependent Proteomics with Split-BioID
315
BSA to decrease nonspecific protein binding. We recommend
not using pre-coated beads, as BSA will later be a major source
of contamination for the MS analysis.
7. We usually prepare lysis and wash buffers freshly from stock
solutions (1 M Tris–HCl, pH 7.4 or 8, 5 M NaCl, 500 mM
EDTA pH 8, 20% SDS, 20% Triton X-100, 20% NP-40, 10%
sodium deoxycholate, 5 M LiCl, 1 M DTT).
8. Low-fluorescence PVDF membranes are required for
fluorescence-based detection of IR dyes-conjugated secondary
antibody. From all the PVDF membranes we tested, only
Immobilon
® -FL PVDF membrane from Millipore gave satisfactory results. Alternatively nitrocellulose membranes typically
yield low autofluorescence. If using chemoluminescence-based
detection, any immunoblotting membrane is fine. Detection of
biotinylated proteins with IR dye- or HRP-conjugated streptavidin works equally well.
9. Ultrafast transfer protocols (7 min) allowed by the Transblot
Turbo system from Bio-Rad or equivalent devices is sufficient
for the detection of biotinylated proteins with conjugated
streptavidin. Any other transfer method is also fine and may
yield better results when the detection of the fusion proteins is
needed, especially for large-size proteins.
10. The use of precast gels and commercial reagents such as loading buffer is optional but recommended to avoid excessive
keratin contamination when running SDS-PAGE for MS
samples.
11. We have provided split-BioID plasmids (see https://www.
addgene.org/Julien_Bethune/) that allow the bidirectional
expression of the NBirA* and CBirA* fusions in various orientations and under the control of the same tet-responsive element [8]. Of important note, these plasmids should be used in
tet-on or tet-off cell lines. The two fusion proteins can be
expressed from the same plasmid or from two separate plasmids. When testing a pair of protein for the first time, we
recommend trying all possible iterations of the fusion proteins
(NBirA* and CBirA* appended at the N- or C- terminus of
each protein of interest, and protein 1 appended to NBirA* or
CBirA*). We did observe that certain combination work significantly better than others.
12. If later many of the resulting isolated plasmids prove to be the
wrong constructs, a dephosphorylation can be included. Typically we digest the plasmids as indicated, and after 1 h, add
alkaline phosphatase buffer and enzyme and let the reaction
Context-Specific Proximity-Dependent Proteomics with Split-BioID
315