229
supplemented with 10% FBS, 2 mM GlutaMAX, and 100 U/
ml of each penicillin and streptomycin.
3. PC12 Tet-Off cells, assay medium: DMEM (1 g glucose/l,
without l-glutamine and without phenol red), 500 ml, supplemented with 1% FBS, and 2 mM GlutaMAX.
4. PC12 Tet-Off cells, medium for maintenance: DMEM (1 g
glucose/l, without L-glutamine, with phenol red), 500 ml,
supplemented with 5% FBS and 10% HS, 2 mM GlutaMAX,
and 100 U/ml of each penicillin and streptomycin.
3 Methods
1. Design appropriate ORF(X)-NTEV-tevS-GV and ORF(Y)CTEV fusion expression plasmids (see guidelines in Subheading
2.1, items 1 and 2, and Note 2). When testing interactions
between candidates X and Y, it is strongly advisable to design
appropriate interaction controls (see Note 5).
2. Clone and prepare sufficient amount of DNA for the assays, set
the concentration for each plasmid to 100 ng/μl (see Note 6).
3. Prepare 96-well plates for split TEV luciferase assays and fluorescence controls.
(a) Coat a white/clear 96-well plate(s) with a 0.02 mg/ml
poly-L lysine (PLL) solution for 30 min at room
temperature.
(b) Wash twice with double deionized water, then let plates
air-dry.
PLL-coated plates can be stored at 4 °C for up to 6 weeks
without losing coating efficiency. This step is optional for
HEK293 cells.
4. Seed 20,000 HEK cells resuspended in maintenance medium
without antibiotics into each well of the coated 96-well plate.
Place the 96-well plate(s) into the incubator. For PC12 TetOff cells, seed 40,000 cells per well (see Note 7).
5. Seed cells onto a clear 96-well plate for an optical control of
transfection efficiency.
6. On the next day, prepare the plasmid mixes for each condition
to be tested. Run 6 replicates per condition to obtain sufficient
readings for statistical analyses. For each 96-well, use 15 ng per
single DNA. Make a master mix for 6 + 1 replicates, which
contains an extra amount needed to allow for pipetting errors
(for example, when using 15 ng DNA/well, use 15 ng x 7 =
105 ng in total). For the Renilla luciferase plasmid, use the
same DNA amount chosen. Prepare a constitutively expressing
GFP plasmid (or variant thereof) (2 ng per well) for control
Characterising Dynamic PPIs Using Split TEV
supplemented with 10% FBS, 2 mM GlutaMAX, and 100 U/
ml of each penicillin and streptomycin.
3. PC12 Tet-Off cells, assay medium: DMEM (1 g glucose/l,
without l-glutamine and without phenol red), 500 ml, supplemented with 1% FBS, and 2 mM GlutaMAX.
4. PC12 Tet-Off cells, medium for maintenance: DMEM (1 g
glucose/l, without L-glutamine, with phenol red), 500 ml,
supplemented with 5% FBS and 10% HS, 2 mM GlutaMAX,
and 100 U/ml of each penicillin and streptomycin.
3 Methods
1. Design appropriate ORF(X)-NTEV-tevS-GV and ORF(Y)CTEV fusion expression plasmids (see guidelines in Subheading
2.1, items 1 and 2, and Note 2). When testing interactions
between candidates X and Y, it is strongly advisable to design
appropriate interaction controls (see Note 5).
2. Clone and prepare sufficient amount of DNA for the assays, set
the concentration for each plasmid to 100 ng/μl (see Note 6).
3. Prepare 96-well plates for split TEV luciferase assays and fluorescence controls.
(a) Coat a white/clear 96-well plate(s) with a 0.02 mg/ml
poly-L lysine (PLL) solution for 30 min at room
temperature.
(b) Wash twice with double deionized water, then let plates
air-dry.
PLL-coated plates can be stored at 4 °C for up to 6 weeks
without losing coating efficiency. This step is optional for
HEK293 cells.
4. Seed 20,000 HEK cells resuspended in maintenance medium
without antibiotics into each well of the coated 96-well plate.
Place the 96-well plate(s) into the incubator. For PC12 TetOff cells, seed 40,000 cells per well (see Note 7).
5. Seed cells onto a clear 96-well plate for an optical control of
transfection efficiency.
6. On the next day, prepare the plasmid mixes for each condition
to be tested. Run 6 replicates per condition to obtain sufficient
readings for statistical analyses. For each 96-well, use 15 ng per
single DNA. Make a master mix for 6 + 1 replicates, which
contains an extra amount needed to allow for pipetting errors
(for example, when using 15 ng DNA/well, use 15 ng x 7 =
105 ng in total). For the Renilla luciferase plasmid, use the
same DNA amount chosen. Prepare a constitutively expressing
GFP plasmid (or variant thereof) (2 ng per well) for control
Characterising Dynamic PPIs Using Split TEV