3.1.3 Mammalian Stable
Cell Lines
In order to generate a stable cell line expressing the desired FLAGtagged protein as in the case of TANC1:
1. Transfect HEK293T cells as described above (see Note 4).
2. Forty-eight hours after transfection, rinse the cells once with
PBS and dissociate with 1mL of trypsin–EDTA.
3. Resuspend trypsinized cells in 10 mL of DMEM cell growth
media.
4. Half of the resuspended cells are transferred in a 15-cm petri
dish containing 20 mL of DMEM–FBS medium supplemented
with 2 μg/mL of puromycin (see Note 5).
5. Stable cell line is then isolated by replacing the selective media
every 2–3 days or more often depending on the rate of cell
death observed.
3.1.4 Characterization
of Stable Cell Lines
Polyclonal stable cell lines can be obtained within 2 weeks of
selection. Clonal selection can take up to 4 weeks due to the
isolation of unique cells in 96-well plate and subsequent clonal
amplification. Immediately after obtaining the stable cell lines,
carry out the following steps:
1. Prepare frozen vials and store the cells adequately.
2. Regularly reassess the level of expression of the fusion proteins,
particularly before large-scale purification.
3. Subcellular localization of the constructs can also be checked
by immunostaining and confocal imaging to confirm the correct localization of the given FLAG-tagged protein of interest.
For example, TANC1 has a cytosolic localization when
expressed in HEK293T cells (Fig. 2b).
3.1.5 Amplification
of Expressing Cells
for Large-Scale Purification
To obtain the adequate quantity of protein complexes for successful
detection by MS, it is recommended to have relatively large amount
of starting material.
1. Start the expansion of stably transfected cells by transferring
one confluent 10-cm cell culture Petri-dish into two 15-cm
plates.
2. Then expand to ten 15-cm plates. More plates may be used if
necessary (see Note 6).
3. In parallel, prepare the same quantity of HEK293T cells stably
transfected with an empty pIRES-puro vector and process
them in a similar way throughout the purification procedure.
3.1.6 Preparation of Cell
Extracts
and Immunopurification
Although different procedures can be applied to harvest the cells,
for HEK293T cells, we use the following procedure. All procedures
are performed at 4
C and all buffers are prechilled on ice (see Notes
7 and 8).
22
Avais M. Daulat et al.
Cell Lines
In order to generate a stable cell line expressing the desired FLAGtagged protein as in the case of TANC1:
1. Transfect HEK293T cells as described above (see Note 4).
2. Forty-eight hours after transfection, rinse the cells once with
PBS and dissociate with 1mL of trypsin–EDTA.
3. Resuspend trypsinized cells in 10 mL of DMEM cell growth
media.
4. Half of the resuspended cells are transferred in a 15-cm petri
dish containing 20 mL of DMEM–FBS medium supplemented
with 2 μg/mL of puromycin (see Note 5).
5. Stable cell line is then isolated by replacing the selective media
every 2–3 days or more often depending on the rate of cell
death observed.
3.1.4 Characterization
of Stable Cell Lines
Polyclonal stable cell lines can be obtained within 2 weeks of
selection. Clonal selection can take up to 4 weeks due to the
isolation of unique cells in 96-well plate and subsequent clonal
amplification. Immediately after obtaining the stable cell lines,
carry out the following steps:
1. Prepare frozen vials and store the cells adequately.
2. Regularly reassess the level of expression of the fusion proteins,
particularly before large-scale purification.
3. Subcellular localization of the constructs can also be checked
by immunostaining and confocal imaging to confirm the correct localization of the given FLAG-tagged protein of interest.
For example, TANC1 has a cytosolic localization when
expressed in HEK293T cells (Fig. 2b).
3.1.5 Amplification
of Expressing Cells
for Large-Scale Purification
To obtain the adequate quantity of protein complexes for successful
detection by MS, it is recommended to have relatively large amount
of starting material.
1. Start the expansion of stably transfected cells by transferring
one confluent 10-cm cell culture Petri-dish into two 15-cm
plates.
2. Then expand to ten 15-cm plates. More plates may be used if
necessary (see Note 6).
3. In parallel, prepare the same quantity of HEK293T cells stably
transfected with an empty pIRES-puro vector and process
them in a similar way throughout the purification procedure.
3.1.6 Preparation of Cell
Extracts
and Immunopurification
Although different procedures can be applied to harvest the cells,
for HEK293T cells, we use the following procedure. All procedures
are performed at 4
C and all buffers are prechilled on ice (see Notes
7 and 8).
22
Avais M. Daulat et al.