AlphaLISA SureFire Ultra Human Phospho-MERTK (Tyr749) Detection Kit, 500 Assay Points

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 permits the cells' 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 AlphaLISA SureFire Ultra quality.

Activation of MERTK Phospho (Tyr749) in cells treated with Gas6 protein

PANC-1 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 Gas6 protein for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MERTK Phospho (Tyr749) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were 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, the Gas6 protein triggered a dose-dependent increase in levels of Phospho (Tyr749) while MERTK Total remained unchanged.

Activation of MERTK Phospho (Tyr749) in primary macrophages

PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiated them into macrophages. Macrophages were seeded in a 96-well plate (30,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO₂.  The cells were then treated with 200 nM MERTK Agonist Antibody for 15 minutes.

After treatment, the cells were lysed with 100 µL of lysis buffer for 10 minutes at RT with shaking (350 rpm). MERTK Phospho (Tyr749) and Total levels were evaluated using AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 3,000 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, the Agonist Antibody increased MERTK (Tyr749) phosphorylation levels by approximately 26-fold. Total protein levels decreased approximately 2-fold, most likely due to Agonist Antibody induced internalization of MERTK.

PANC-1 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 left untreated or treated with 200 nM MERTK Agonist Antibody for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MERTK Phospho (Tyr749) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were 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, the Agonist Antibody increased MERTK (Tyr749) phosphorylation levels by approximately 24-fold. Total protein levels decreased approximately 2-fold, most likely due to Agonist Antibody induced internalization of MERTK.

THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in complete medium containing 100 nM PMA, and incubated for 24 hours at 37°C, 5% CO₂. The cells were starved in serum free medium for 24 hours then left untreated or treated with 200 nM MERTK Agonist Antibody for 15 minutes.

After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MERTK Phospho (Tyr749) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 10,000 cells) were 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, the Agonist Antibody increased MERTK (Tyr749) phosphorylation levels by approximately 132-fold. Total protein levels decreased approximately 2-fold, most likely due to Agonist Antibody induced internalization of MERTK.

Inhibition of MERTK Tyr749 phosphorylation upon treatment with UNIC1062 inhibitor

Hep G2 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were pretreated with 100 µM Pervanadate for 15 minutes, then cells were left untreated or treated with 5 µM UNC1062 (MERTK-selective tyrosine kinase inhibitor) for 1 hour.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MERTK Phospho (Tyr749) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 5,000 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, UNC1062 inhibited pervanadate-induced MERTK Tyr749 phosphorylation while Total levels remained unchanged.

Knockout validation of MERTK Phospho (Tyr749) assay

MERTK Phospho (Tyr749) levels were assessed in Wild Type (WT) and MERTK knockout (KO) A549 cells. MERTK KO cells (Abcam ab301074) and WT A549 cells were seeded in a T75 flask (6 x 10⁶ cells/flask) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were treated with 100 µM Pervanadate for 30 minutes.

After treatment, the cells were lysed with 2 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). The cell lysate was serially diluted with Lysis Buffer and MERTK Phospho (Tyr749) levels were then evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate 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, MERTK Phospho (Tyr749) was detected in the pervanadate treated WT cells but not in the MERTK-KO cell line.