IDG Kinase - NanoBRET Assays

NanoBRET Assay Protocol

NanoBRET transient transfection (general)

For cellular NanoBRET Target Engagement experiments, the NL-kinase fusion construct was diluted with carrier DNA - pGEM-3Zf(-) (Promega, Madison, WI, USA) at a mass ratio of 1:10 (mass/mass), prior to adding FuGENE HD (Promega, Madison, WI, USA). DNA:FuGENE complexes were formed at a ratio of 1:3 (mg DNA/mL FuGENE HD) according to the manufacturer’s protocol (Promega, Madison, WI, USA). The resulting transfection complex (1 part, volume) was then gently mixed with 20 parts (v/v) of HEK-293 cells (ATCC) suspended at a density of 2 x 105 cells/mL in DMEM (Gibco) + 10% FBS (Seradigm/VWR). The cells were then plated into Tissue Culture Treated, 96-well plates (Corning) followed by incubation (37 °C/ 5 % CO2) for 24 hours. After 24 hours the medium was removed and to each well was added 85 μL of Opti-MEM media (Gibco).

NanoBRET transient transfection (for CDK10-CDK20)

For cellular NanoBRET Target Engagement experiments, the NL-kinase fusion construct was diluted with appropriate cyclin DNA - pGEM-3Zf(-) (Promega, Madison, WI, USA) at a mass ratio of 1:10 (mass/mass), prior to adding FuGENE HD (Promega, Madison, WI, USA). DNA:FuGENE complexes were formed at a ratio of 1:3 (mg DNA/mL FuGENE HD) according to the manufacturer’s protocol (Promega, Madison, WI, USA). The resulting transfection complex (1 part, volume) was then gently mixed with 20 parts (v/v) of HEK-293 cells (ATCC) suspended at a density of 2 x 105 cells/mL in DMEM (Gibco) + 10% FBS (Seradigm/VWR). The cells were then plated into Tissue Culture Treated, 96-well plates (Corning) followed by incubation (37 °C/ 5 % CO2) for 24 hours. After 24 hours the medium was removed and to each well was added 85 μL of Opti-MEM media (Gibco).

NanoBRET assay protocol

Utilizing the provided recommended Tracer concentration (see tracer titration sheets) add 5 μLof 20x working stock of NanoBRET Tracer to all wells, except the “no tracer” control wells. All inhibitors were prepared initially as concentrated stock solutions in 100% DMSO (Sigma). A total of 10 μL/well of the 10x chemical inhibitor stock solutions (final assay concentration 1 % DMSO) were added. For “no compound” and “no tracer” control wells, a total of 10 μL/well of Opti-MEM plus DMSO (9 μL was added (final concentration 1 % DMSO)). 96 well plates containing cells with the appropriate nanoBRET Tracer and inhibitors (100 μL total volume per well) were equilibrated (37 °C/ 5 % CO2) for 2 hours. To measure nanoBRET signal, nanoBRET NanoGlo substrate at a ratio of 1:166 to Opti-MEM media in combination with extracellular NanoLuc Inhibitor diluted 1:500 (10 μL [30 mM stock] per 5 mL Opti-MEM plus substrate) were combined to create a 3x stock. A total of 50 μL of the 3x substrate/extracellular NanoLuc inhibitor were added to each well. The plates were read within 15 minutes (GloMax Discover luminometer, Promega, Madison, WI, USA) equipped with 450 nM BP filter (donor) and 600 nM LP filter (acceptor), using 0.3 s integration time instrument utilizing the “nanoBRET 618” protocol.

Prior to curve fitting, the average BRET ratio for “no tracer” (Opti-MEM + DMSO only) wells was subtracted from all experimental inhibitor well BRET ratio values and converted to mBRET units (x1000). Additional normalization of the NanoBRET assay data was performed by converting experimental values for respective concentrations of experimental inhibitors to relative percent control values (no compound [Opti-MEM + DMSO + Tracer 5 only] wells = 100 % Control, no tracer [Opti-MEM + DMSO] wells = 0 % Control). The data was normalized to 0 % and 100 % inhibition control values and fitted to a four parameter dose-response binding curve in GraphPad Software (version 7, La Jolla, CA, USA).

NanoBRET tracer competition in transiently transfected cells

HEK293 cells transiently expressing kinase-NanoLuc fusion protein were resuspended in assay Medium, seeded into 96-well plates and mixed with various concentrations of the appropriate NanoBRET Tracer. Cells were treated with varying concentrations of unlabeled compound as a competitive inhibitor for 2 hours before adding 3X Complete Substrate plus Inhibitor Solution. BRET was measured using a GloMax Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted vs. unmodified test compound concentration to determine apparent intracellular affinity of the unmodified test compound at each concentration of tracer.

*Note that lower tracer concentrations result in more accurate estimation of intracellular compound affinity but a lower assay window. The use of a lower concentration of tracer may be more accurate when quantifying intracellular compound affinity.

NanoBRET target engagement assay in transiently transfected cells

HEK293 cells expressing kinase-NanoLuc fusion protein were resuspended in assay Medium, seeded into 96-well plates and mixed with various concentrations of the appropriate NanoBRET Tracer. Cells were treated with tracer only or an excess of unlabeled compound (10 μM) as a competitive inhibitor for 2 hours before adding 3X Complete Substrate plus Inhibitor Solution. BRET was measured using a GloMax Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted vs. unmodified test compound concentration to determine apparent intracellular affinity of the unmodified test compound at each concentration of tracer.

  1. Vasta, J. D.; Corona, C. R.; Wilkinson, J.; Zimprich, C. A.; Hartnett, J. R.; Ingold, M. R.; Zimmerman, K.; Machleidt, T.; Kirkland, T. A.; Huwiler, K. G.; Ohana, R. F.; Slater, M.; Otto, P.; Cong, M.; Wells, C. I.; Berger, B. T.; Hanke, T.; Glas, C.; Ding, K.; Drewry, D. H.; Huber, K. V. M.; Willson, T. M.; Knapp, S.; Muller, S.; Meisenheimer, P. L.; Fan, F.; Wood, K. V.; Robers, M. B., Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell Chem Biol 2018, 25 (2), 206-214 e11.

* Please inquire about this material by emailing Promega at CAS@promega.com.

* Additional inquires can be to CAS@promega.com or techserv@promega.com


NanoBRET Assay List
Kinase Kinase Fusion Name Kinase Fusion Catalog Number Tracer Name Tracer Catalog Number Tracer Concentration Additional Tracer Information
NEK1 NanoLuc®-NEK1 NV3761 NA NA NA NA
NEK6 NEK6-NanoLuc® NV3801 NA NA NA Here
NIM1K NIM1K-NanoLuc® NV3811 NA NA NA Here
NRK NanoLuc®-NRK NV3831 NA NA NA NA
PAK6 PAK6-NanoLuc® NV3861 NA NA NA Here
PIP5K1B NanoLuc®-PIP5K1B NV4071 NA NA NA NA
STK36 NanoLuc®-STK36 NV4331 NA NA NA NA
TESK1 NanoLuc®-TESK1 NV2161 NA NA NA Here
CDK10 NanoLuc®-fused CDK10 Special Request 7281 NA 0.25 µM Here
CDK11A NanoLuc®-fused CDK11A Special Request 7281 NA 0.125 µM Here
CDK11B NanoLuc®-fused CDK11B Special Request 7281 NA 0.125 µM Here
CDK12 NanoLuc®-fused CDK12 Special Request 7281 NA 0.5 µM Here
CDK13 NanoLuc®-fused CDK13 Special Request 7281 NA 0.5 µM Here
CDK14 NanoLuc®-fused CDK14 Special Request 7281 NA 0.125 µM Here
CDK16 NanoLuc®-fused CDK16 Special Request 7281 NA 0.125 µM Here
CDK17 NanoLuc®-fused CDK17 Special Request 7281 NA 0.125 µM Here
CDK18 NanoLuc®-fused CDK18 Special Request 7281 NA 0.125 µM Here
CLK4 NanoLuc®-fused CLK4 Special Request K-5 N248A 0.5 µM Here
CSNK2A2 NanoLuc®-fused CSNK2A2 Special Request K-5 N248A 1 µM Here
DYRK2 NanoLuc®-fused DYRK2 Special Request K-5 N248A 1 µM Here
LTK NanoLuc®-fused LTK Special Request K-5 N248A 1 µM Here
BRSK2 NanoLuc®-fused BRSK2 Special Request K-5 N248A 1 µM Here
CDK15 NanoLuc®-fused CDK15 Special Request 7281 NA 0.063 µM Here
CDK20 NanoLuc®-fused CDK20 Special Request K-9/7520 NA 0.5 µM Here
CDKL1 NanoLuc®-fused CDKL1 Special Request 6908 NA 0.25 µM Here
CDKL2 NanoLuc®-fused CDKL2 Special Request 6908 NA 0.25 µM Here
CDKL3 NanoLuc®-fused CDKL3 Special Request 6908 NA 0.25 µM Here
CDKL5 NanoLuc®-fused CDKL5 Special Request 6908 NA 0.25 µM Here
CSNK1G2 NanoLuc®-fused CSNK1G2 Special Request K-5 N248A 1 µM Here
DYRK1B NanoLuc®-fused DYRK1B Special Request K-5 N248A 1 µM Here
MKNK2 NanoLuc®-fused MKNK2 Special Request K-10 7600 NA 0.5 µM Here
NEK3 NanoLuc®-fused NEK3 Special Request K-5 N248A 1 µM Here
NEK9 NanoLuc®-fused NEK9 Special Request K-5 N248A 1 µM Here
RIOK2 NanoLuc®-fused RIOK2 Special Request K-5 N248A 1 µM Here
STK33 NanoLuc®-fused STK33 Special Request K-5 N248A 1 µM Here
PAK5 NanoLuc®-fused PAK5 Special Request K-5 N248A 1 µM Here
PKN3 NanoLuc®-fused PKN3 Special Request K-5 N248A 1 µM Here
STK17B NanoLuc®-fused STK17B Special Request 7227 NA 0.25 µM Here
CDK19 NanoLuc®-fused CDK19 Special Request 8/7235 NA 0.063 µM Here
DCLK3 NanoLuc®-fused DCLK3 Special Request 10-7600 NA 0.5 µM Here
HIPK1 NanoLuc®-fused HIPK1 Special Request 10-7600 NA 0.5 µM Here
HIPK3 NanoLuc®-fused HIPK3 Special Request 10-7600 NA 0.5 µM Here
HIPK4 NanoLuc®-fused HIPK4 Special Request 10-7600 NA 0.5 µM Here
MAPK4 NanoLuc®-fused MAPK4 Special Request K-5 N248A 1 µM Here
MARK4 NanoLuc®-fused MARK4 Special Request K-5 N248A 1 µM Here
NEK4 NanoLuc®-fused NEK4 Special Request 10-7600 NA 0.5 µM Here
NEK11 NanoLuc®-fused NEK11 Special Request 10-7600 NA 0.5 µM Here
CAMK1D NanoLuc®-fused CAMK1D Special Request 9-7520 NA 0.5 µM Here
ICK NanoLuc®-fused ICK Special Request 12-7281 NA 0.25 µM Here
MAP3K10 NanoLuc®-fused MAP3K10 Special Request K-5 N248A 1 µM Here
MARK3 NanoLuc®-fused MARK3 Special Request 10-7600 NA 0.5 µM Here
NEK5 NanoLuc®-fused NEK5 Special Request 10-7600 NA 0.0625 µM Here
PHKG1 NanoLuc®-fused PHKG1 Special Request K-5 N248A 1 µM Here
STK3 NanoLuc®-fused STK3 Special Request 10-7600 NA 1 µM Here
STK32A NanoLuc®-fused STK32A Special Request 9-7520 NA 0.5 µM Here
STK32B NanoLuc®-fused STK32B Special Request K-5 N248A 0.5 µM Here
STK38L NanoLuc®-fused STK38L Special Request 10-7600 NA 0.5 µM Here
DYRK3 NanoLuc®-fused DYRK3 Special Request K-5 N248A 1 µM Here
ERN2 NanoLuc®-fused ERN2 Special Request 10-7600 NA 0.5 µM Here
MAP3K21 NanoLuc®-fused MAP3K21 Special Request 10-7600 NA 0.5 µM Here
COQ8B NanoLuc®-fused COQ8B Special Request 10-7600 NA 0.5 µM Here
CSNK1A1L NanoLuc®-fused CSNK1A1L Special Request 10-7600 NA 0.5 µM Here
DSTYK NanoLuc®-fused DSTYK Special Request 10-7600 NA 0.5 µM Here
MAST3 NanoLuc®-fused MAST3 Special Request 10-7600 NA 0.5 µM Here
MAST4 NanoLuc®-fused MAST4 Special Request 10-7600 NA 0.5 µM Here
PHKG2 NanoLuc®-fused PHKG2 Special Request 10-7600 NA 1 µM Here
SBK3 NanoLuc®-fused SBK3 Special Request K-5 N248A 1 µM Here
WEE2 NanoLuc®-fused WEE2 Special Request K-5 N248A 0.5 µM Here
NUAK2 NanoLuc®-fused NUAK2 Special Request 10-7600 NA 0.5 µM Here
PKMYT1 NanoLuc®-fused PKMYT1 Special Request K-5 N248A 1 µM Here
PRKACB NanoLuc®-fused PRKACB Special Request 10-7600 NA 0.5 µM Here
STK17A NanoLuc®-fused STK17A Special Request 10-7600 NA 0.5 µM Here
TLK1 NanoLuc®-fused TLK1 Special Request 10-7600 NA 0.5 µM Here
TLK2 NanoLuc®-fused TLK2 Special Request 10-7600 NA 0.5 µM Here
PIP4K2C NanoLuc®-fused PIP4K2C Special Request 8-7235 NA 0.25 µM Here
TSSK1B NanoLuc®-fused TSSK1B Special Request 9-7520 NA 0.5 µM Here
CAMK1G NanoLuc®-fused CAMK1G Special Request 9-7520 NA 0.5 µM Here
BRSK1 NanoLuc®-fused BRSK1 Special Request 10-7600 NA 0.5 µM Here
NIM1K NanoLuc®-fused NIM1K Special Request K-10 NA 0.5 µM Here
CSNK1G1 NanoLuc®-fused CSNK1G1 Special Request 12-7281 NA 0.4 µM Here
CSNK1G3 NanoLuc®-fused CSNK1G3 Special Request 12-7281 NA 0.4 µM Here
NEK6 NanoLuc®-fused NEK6 Special Request K9 NA 0.17 µM Here
PAK6 NanoLuc®-fused PAK6 Special Request K-10 NA 0.5 µM Here
TESK1 NanoLuc®-fused TESK1 Special Request K4 NA 0.5 µM Here
TTBK2 NanoLuc®-fused TTBK2 Special Request Tracer 29 NA 2 µM Here
TTBK1 NanoLuc®-fused TTBK1 Special Request Tracer 29 NA 2 µM Here
PIP5K1C NanoLuc®-fused PIP5K1C Special Request Tracer FP8-39-2 NA 7.8 µM Here