2. On the next day, remove the medium from the dishes and
replace with 10 mL of fresh medium.
3. Prepare the transfection mixes for all conditions to be tested,
usually the pair of proteins of interest and a negative control (see
Note 15). When using PEI as a transfection reagent for HeLa
cells, we typically use 12 μg PEI diluted in 150 μL DMEM
medium (without serum) mixed with 6 μg plasmid diluted in
150 μL DMEM medium (without serum) per plate. The transfection mixes are incubated for 5–15 min at room temperature
(RT) and then dispatched drop wise to their corresponding
dishes (300 μL per 10 cm plate). The cells are then incubated
overnight at 37
C, 5% CO 2 .
4. On the next day, transfer the cells from each dish to 15 cm
plates. To do so, the cells are first washed with 7 mL PBS and
then incubated with 1.5 mL of a trypsin-EDTA solution for
5 min at RT. DMEM (3.5 mL) is then added and the cells
resuspended by pipetting and transferred to 15 cm plates filled
with 20 mL DMEM containing enough biotin to reach a final
concentration of 50 μM for a total volume of 25 mL, and
enough doxycycline for a concentration of 200 ng mL
À1 doxycycline if using inducible plasmids under the control of a
tet-responsive element (see Note 16). Incubate the cells for at
least 16 h at 37
C, 5% CO 2 .
5. On the following day, cells in each 15 cm dish are washed twice
with 20 mL PBS and then scrapped in 1.5 mL PBS using
disposable cell scrappers.
6. Scrapped cells are then transferred to 1.5 mL tubes and harvested by centrifugation at 1200 Â g for 5 min at 4
C.
7. Cell pellets can then be snap frozen in liquid nitrogen and if
desired stored at À80
C until further processing.
8. To prepare cell lysates, the frozen cell pellets are first resuspended in 1 mL lysis buffer II at room temperature and passed
ten times (five strokes) through 25G needle attached to a 1-mL
syringe (see Note 21).
9. The lysates are then sonicated in a cold ultrasonic bath for
4 cycles of 30 s at high intensity with 30 s pause time between
each cycle (see Note 22).
10. Add Triton X-100 to the sonicated lysates to a final concentration of 2% (Typically 100 μL of a 20% Triton X-100 solution is
added to 900 μL of lysate) and then enough volume of 50 mM
Tris–HCl, pH 7.4 solution, so that the final NaCl concentration is adjusted to 150 mM (typically 2.3 mL is added to 1 mL
of Triton X-100 adjusted lysates) (see Note 23).
Context-Specific Proximity-Dependent Proteomics with Split-BioID
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replace with 10 mL of fresh medium.
3. Prepare the transfection mixes for all conditions to be tested,
usually the pair of proteins of interest and a negative control (see
Note 15). When using PEI as a transfection reagent for HeLa
cells, we typically use 12 μg PEI diluted in 150 μL DMEM
medium (without serum) mixed with 6 μg plasmid diluted in
150 μL DMEM medium (without serum) per plate. The transfection mixes are incubated for 5–15 min at room temperature
(RT) and then dispatched drop wise to their corresponding
dishes (300 μL per 10 cm plate). The cells are then incubated
overnight at 37
C, 5% CO 2 .
4. On the next day, transfer the cells from each dish to 15 cm
plates. To do so, the cells are first washed with 7 mL PBS and
then incubated with 1.5 mL of a trypsin-EDTA solution for
5 min at RT. DMEM (3.5 mL) is then added and the cells
resuspended by pipetting and transferred to 15 cm plates filled
with 20 mL DMEM containing enough biotin to reach a final
concentration of 50 μM for a total volume of 25 mL, and
enough doxycycline for a concentration of 200 ng mL
À1 doxycycline if using inducible plasmids under the control of a
tet-responsive element (see Note 16). Incubate the cells for at
least 16 h at 37
C, 5% CO 2 .
5. On the following day, cells in each 15 cm dish are washed twice
with 20 mL PBS and then scrapped in 1.5 mL PBS using
disposable cell scrappers.
6. Scrapped cells are then transferred to 1.5 mL tubes and harvested by centrifugation at 1200 Â g for 5 min at 4
C.
7. Cell pellets can then be snap frozen in liquid nitrogen and if
desired stored at À80
C until further processing.
8. To prepare cell lysates, the frozen cell pellets are first resuspended in 1 mL lysis buffer II at room temperature and passed
ten times (five strokes) through 25G needle attached to a 1-mL
syringe (see Note 21).
9. The lysates are then sonicated in a cold ultrasonic bath for
4 cycles of 30 s at high intensity with 30 s pause time between
each cycle (see Note 22).
10. Add Triton X-100 to the sonicated lysates to a final concentration of 2% (Typically 100 μL of a 20% Triton X-100 solution is
added to 900 μL of lysate) and then enough volume of 50 mM
Tris–HCl, pH 7.4 solution, so that the final NaCl concentration is adjusted to 150 mM (typically 2.3 mL is added to 1 mL
of Triton X-100 adjusted lysates) (see Note 23).
Context-Specific Proximity-Dependent Proteomics with Split-BioID
311