changes in binding affinity can be monitored by ITC
measurements [9].
1. Add 0.2 ml of PDZ stock solution to 1.8 ml Buffer C. Dialyze
against Buffer C together with the RSK recombinant fragment
stock solution at 4
C overnight.
2. Determine the PDZ and the unlabeled RSK recombinant fragment concentrations (see Note 7). Alternatively, the lyophilized
peptide can be dissolved in buffer C.
3. Fill the ITC cell with the PDZ domain solution (see Note 9).
4. Fill the ITC syringe with the RSK recombinant fragment solution (see Note 9).
5. Wait for the equilibration of temperature between the reference and sample cells (until the difference power known as DP
reach close to zero), then run the ITC program (see Note 10).
6. Analyze data using the built-in model “one set of sites” in
Origin for ITC software. For more information, check the
following reference [9].
3.4 NanoBiT Protein–
Protein Interaction
Assay to Study
the Effects
of Phosphorylation
NanoBiT is a split-luciferase fragment complementation assay
(Fig. 3), which is appropriate for monitoring dynamic changes in
PPIs [25]. Instead of using isolated fragments and peptides of PDZ
domain and PBM containing proteins, the interaction can be studied with full length proteins in a cellular environment. The advantage of the NanoBiT system over other bimolecular fragment
complementation assays is the low, almost endogenous protein
expression due to the HSV promoter and the reversible interaction
between the split NanoLuc fragment that makes possible to monitor dynamic changes in signaling pathways beside the steady-state
signal [9]. The association and dissociation of the PDZ–RSK1
complexes can be investigated under EGF stimulation conditions,
which allows one to characterize the effect of phosphorylation on
PBMs.
1. Plate approximately 20,000 HEK293T cells/well in a white
TC-treated 96-well plate by distributing 100 μl of a 200,000
cell/ml cell suspension in six parallel wells per measurement
(see Note 11). For measurements at 37
C, add 100 μl sterile
PBS to the outside wells and spaces between the inner wells.
2. Incubate the plate at 37
C with 5% CO 2 for 16–24 h.
3. Dilute the RSK1-SmBiT and PDZ-LgBiT plasmid DNAs to
150 ng/μl.
4. 4. Pipet 52.44 μl of Opti-MEM I buffer to a sterile Eppendorf
tube per measurement, add 2.52 μl of each of the SmBiT and
LgBiT plasmids, then add 2.52 μl FuGene HD reagent and mix
it by pipetting (see Note 12).
Regulation of RSK1-PDZ Domain Interactions
187
measurements [9].
1. Add 0.2 ml of PDZ stock solution to 1.8 ml Buffer C. Dialyze
against Buffer C together with the RSK recombinant fragment
stock solution at 4
C overnight.
2. Determine the PDZ and the unlabeled RSK recombinant fragment concentrations (see Note 7). Alternatively, the lyophilized
peptide can be dissolved in buffer C.
3. Fill the ITC cell with the PDZ domain solution (see Note 9).
4. Fill the ITC syringe with the RSK recombinant fragment solution (see Note 9).
5. Wait for the equilibration of temperature between the reference and sample cells (until the difference power known as DP
reach close to zero), then run the ITC program (see Note 10).
6. Analyze data using the built-in model “one set of sites” in
Origin for ITC software. For more information, check the
following reference [9].
3.4 NanoBiT Protein–
Protein Interaction
Assay to Study
the Effects
of Phosphorylation
NanoBiT is a split-luciferase fragment complementation assay
(Fig. 3), which is appropriate for monitoring dynamic changes in
PPIs [25]. Instead of using isolated fragments and peptides of PDZ
domain and PBM containing proteins, the interaction can be studied with full length proteins in a cellular environment. The advantage of the NanoBiT system over other bimolecular fragment
complementation assays is the low, almost endogenous protein
expression due to the HSV promoter and the reversible interaction
between the split NanoLuc fragment that makes possible to monitor dynamic changes in signaling pathways beside the steady-state
signal [9]. The association and dissociation of the PDZ–RSK1
complexes can be investigated under EGF stimulation conditions,
which allows one to characterize the effect of phosphorylation on
PBMs.
1. Plate approximately 20,000 HEK293T cells/well in a white
TC-treated 96-well plate by distributing 100 μl of a 200,000
cell/ml cell suspension in six parallel wells per measurement
(see Note 11). For measurements at 37
C, add 100 μl sterile
PBS to the outside wells and spaces between the inner wells.
2. Incubate the plate at 37
C with 5% CO 2 for 16–24 h.
3. Dilute the RSK1-SmBiT and PDZ-LgBiT plasmid DNAs to
150 ng/μl.
4. 4. Pipet 52.44 μl of Opti-MEM I buffer to a sterile Eppendorf
tube per measurement, add 2.52 μl of each of the SmBiT and
LgBiT plasmids, then add 2.52 μl FuGene HD reagent and mix
it by pipetting (see Note 12).
Regulation of RSK1-PDZ Domain Interactions
187