The AlphaLISA™ SureFire® Ultra™ p-JNK1/2/3 (Thr183/Tyr185) assay is a sandwich immunoassay for quantitative detection of phospho-JNK1/2/3 (phosphorylated on Thr183/Tyr185) in cellular lysates using Alpha Technology.
In the AlphaLISA™ SureFire® Ultra™ assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure™ tag. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of phosphoprotein present in the sample.
AlphaLISA™ SureFire® Ultra™ kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire® kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
Induction of JNK phosphorylation in various cell types
PBMCs were isolated from healthy donors and seeded at 400,000 cells/well in a 96 well plate. Cells were treated with 10 ng/mL IL-1b for 10 minutes, 1 µg/mL LPS or 1 µM PMA for 30 minutes.
After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JNK1/2/3 Phospho (Thr183/Tyr185) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
JNK1/2/3 Phospho (Thr183/Tyr185) levels were increased by treatment with LPS and PMA but not IL-1b.
PBMCs were isolated from healthy donors and cultured for 7 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM and incubated overnight at 37°C, 5% CO2. The cells were treated with 1 µg/mL LPS for the indicated time points.
After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JNK1/2/3 Phospho (Thr183/Tyr185) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 8,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
The levels of Phospho JNK1/2/3 (Thr183/Tyr185) after LPS treatment peaked at 30 mins and decreased thereafter.
RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with 500 ng/mL LPS at the indicated time points.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JNK1/2/3 Phospho (Thr183/Tyr185) and JNK1/2 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
LPS triggered an increase in the levels of Phospho JNK1/2/3 (Thr183/Tyr185) that was maximal at 15 minutes and decreased thereafter.
Activation of Phospho JNK1/2/3 (Thr183/Tyr185) in HEK293 cells
HEK293 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were serum starved for 2 hours and then treated increasing concentrations of Anisomycin for 30 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho JNK1/2/3 (Thr183/Tyr185) and Total JNK1/2 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Anisomycin induced a dose-dependent increase in Phospho JNK1/2/3 (Thr183/Tyr185) while Total JNK1/2 levels remained unchanged.
HEK293 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of sorbitol for 30 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho JNK1/2/3 (Thr183/Tyr185) levels were evaluated using AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, sorbitol induced a dose-dependent increase in the levels of Phospho JNK1/2/3 (Thr183/Tyr185).
Inhibition of Phospho JNK1/2/3 (Thr183/Tyr185) in oxozeaenol treated cells
HEK293 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of oxozeaenol for 2 hours and then 0.75 M sorbitol for 30 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho JNK1/2/3 (Thr183/Tyr185) and Total JNK1/2 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, oxozeaenol treatment resulted in a dose-dependent decrease in Phospho JNK1/2/3 (Thr183/Tyr185) while Total JNK1/2 levels remained unchanged.
Phospho (Thr183/Tyr185) JNK1/2/3 assay sensitivity - cell lysate dilution
Cell lysate was prepared from HEK293 cells cultured to confluence in T175 flasks in complete medium. Cells were treated with 1 µg/mL Anisomycin for 30 minutes and then lysed in 10 mL of Lysis Buffer.
Lysates were serially diluted in Lysis Buffer and assayed for Phospho (Thr183/Tyr185) JNK1/2/3 and Total JNK1/2 using respective AlphaLISA SureFire Ultra kits. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Approximate number of cells/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect Phospho (Thr183/Tyr185) JNK1/2/3 in less than 500 cells/datapoint.
