AlphaLISA SureFire Ultra Human and Mouse Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Detection Kit, 100 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 Phospho Ezrin/Radixin/Moesin in Calyculin A treated cells

HeLa 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 Calyculin A for 1 hour.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho Ezrin/Radixin/Moesin and Ezrin/Moesin total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, lysate was further diluted in Lysis Buffer and 10 µL of cell lysate (approximately 200 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, Calyculin A triggered a dose-dependent increase of Ezrin/Radixin/Moesin phosphorylation levels while Total Ezrin/Moesin levels remained unchanged.

Decrease of Phospho Ezrin/Radixin/Moesin levels in Staurosporine treated cells

A549 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 Staurosporine for 1 hour.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho Ezrin/Radixin/Moesin and Ezrin/Moesin total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, lysates were further diluted and 10 µL of cell lysate (approximately 1,000 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, Staurosporine triggered a dose-dependent decrease of Ezrin/Radixin/Moesin phosphorylation levels while no significant changes were detected for Ezrin/Moesin total levels.

Versatility of Phospho Ezrin/Radixin/Moesin assay in various cell lines

Human and mouse cell lysates were diluted with Lysis Buffer to represent approximately 10,000 cells/datapoint. Ezrin/Radixin/Moesin Phospho levels were evaluated using the AlphaLISA SureFire Ultra assay kit.

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.

The Phospho Ezrin/Radixin/Moesin assay contains phospho and total antibodies which recognise the phosphorylated forms of these three proteins. As expected, this assay was able to detect endogenous levels of these phospho proteins in a wide range of cell lines.