3. Stain the gel with colloidal Coomassie Brillant Blue G250
staining as follows (see Note 26): wash the gel three times for
10 min with ultrapure water in a 15-cm bacterial dish. Incubate
the gel with Coomassie staining solution for 2–12 h (until
bands for streptavidin and some biotinylated proteins are
clearly visible) at RT. Optionally destain for 1 h at RT with
destaining solution. Rinse the gel twice briefly with ultrapure
water.
4. For each sample, excise the whole lane with a clean scalpel,
excluding the streptavidin band (typically the main visible band
running at approximately 17 kDa), and transfer to 1.5 mL tube
(Fig. 4b).
5. The excised lanes can be sent to a proteomics facility for in-gel
trypsin digestion and MS analysis (see Note 27).
4 Notes
1. We have tested split-BioID in HeLa cells, in principle it should
work in any mammalian cell type and more generally in any cell
type in which BioID has been demonstrated to work.
2. Any standard cell culture medium appropriate for the chosen
cell line is fine. Most media will already contain biotin as it is an
essential vitamin. However, split-BioID like BioID requires the
addition of an excess (50 μM) of exogenous biotin to work.
3. Any transfection reagent known to promote high transfection
efficiency and low toxicity for the chosen cell line is fine.
4. Biotin has a good solubility in ammonium hydroxide or
NaOH. To make a stock solution of biotin, dissolve it at
50 mg mL
À1 (corresponds to approximately 200 mM) in 2 M
ammonium hydroxide or 2 M NaOH. Once biotin is
completely dissolved, dilute this solution to 50 mM biotin in
500 mM Hepes, pH 7.4, then adjust the pH to 7.4 with HCl.
Sterilize by passing through a 0.22-μM filter, then prepare
aliquots in 1.5-mL safe lock tubes and store the resulting
1000Â stock solution at À20
C. We have used up to 2-yearold stock solutions prepared and stored this way without apparent loss of activity.
5. Doxycycline can be dissolved at 10 mg mL
À1 in 70% ethanol
and stored in a screw cap microtube at À20
C in the dark. The
high percentage alcohol ensures that the stock solution is sterile. We have used up to 1-year-old stock solutions prepared and
stored this way without apparent loss of activity.
6. We routinely perform streptavidin pulldowns with magnetic
Dynabeads MyOne Streptavidin C1 beads. Many other types
of beads, including sepharose beads [5] and NeutrAvidin- [7]
314
Cinthia Amaya Ramirez et al.
staining as follows (see Note 26): wash the gel three times for
10 min with ultrapure water in a 15-cm bacterial dish. Incubate
the gel with Coomassie staining solution for 2–12 h (until
bands for streptavidin and some biotinylated proteins are
clearly visible) at RT. Optionally destain for 1 h at RT with
destaining solution. Rinse the gel twice briefly with ultrapure
water.
4. For each sample, excise the whole lane with a clean scalpel,
excluding the streptavidin band (typically the main visible band
running at approximately 17 kDa), and transfer to 1.5 mL tube
(Fig. 4b).
5. The excised lanes can be sent to a proteomics facility for in-gel
trypsin digestion and MS analysis (see Note 27).
4 Notes
1. We have tested split-BioID in HeLa cells, in principle it should
work in any mammalian cell type and more generally in any cell
type in which BioID has been demonstrated to work.
2. Any standard cell culture medium appropriate for the chosen
cell line is fine. Most media will already contain biotin as it is an
essential vitamin. However, split-BioID like BioID requires the
addition of an excess (50 μM) of exogenous biotin to work.
3. Any transfection reagent known to promote high transfection
efficiency and low toxicity for the chosen cell line is fine.
4. Biotin has a good solubility in ammonium hydroxide or
NaOH. To make a stock solution of biotin, dissolve it at
50 mg mL
À1 (corresponds to approximately 200 mM) in 2 M
ammonium hydroxide or 2 M NaOH. Once biotin is
completely dissolved, dilute this solution to 50 mM biotin in
500 mM Hepes, pH 7.4, then adjust the pH to 7.4 with HCl.
Sterilize by passing through a 0.22-μM filter, then prepare
aliquots in 1.5-mL safe lock tubes and store the resulting
1000Â stock solution at À20
C. We have used up to 2-yearold stock solutions prepared and stored this way without apparent loss of activity.
5. Doxycycline can be dissolved at 10 mg mL
À1 in 70% ethanol
and stored in a screw cap microtube at À20
C in the dark. The
high percentage alcohol ensures that the stock solution is sterile. We have used up to 1-year-old stock solutions prepared and
stored this way without apparent loss of activity.
6. We routinely perform streptavidin pulldowns with magnetic
Dynabeads MyOne Streptavidin C1 beads. Many other types
of beads, including sepharose beads [5] and NeutrAvidin- [7]
314
Cinthia Amaya Ramirez et al.