Ezrin, Radixin, and Moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton, maintaining cell shape and facilitating adhesion and motility. These proteins are activated through phosphorylation, which stabilizes their open conformation, enabling interaction with the cytoskeleton. Overexpression of ERM proteins is observed in cancers such as breast and pancreatic cancer, enhancing metastatic potential. Dysregulated ERM proteins also contribute to autoimmune disorders like multiple sclerosis.
The AlphaLISA SureFire Ultra Human and Mouse Total Ezrin/Moesin Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-Ezrin/Moesin in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes 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 target 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 protein present in the sample.
Total-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 Total-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.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total 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.
Validation of Ezrin/Moesin Total assay 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.
Validation of Ezrin/Moesin Total assay 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.
Ezrin/Moesin Total assay cross-reactivity
Specificity of the Ezrin/Moesin Total assay was assessed using Ezrin, Radixin and Moesin recombinant human proteins.
Dilutions of recombinant Ezrin (Abcam, ab132943), Radixin (RayBiotech, 230-30097) and Moesin (RayBiotech, 230-30095) proteins were prepared at a final concentration of 0.5 µg/mL in Lysis Buffer. Ezrin/Moesin Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein preparation was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT in the dark. 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 Ezrin/Moesin Total assay shows reactivity against Ezrin and Moesin recombinant proteins but does not detect Radixin.
Versatility of Ezrin/Moesin Total assay in various cell lines
Human and mouse cell lysates were diluted with Lysis Buffer to represent approximately 10,000 cells/datapoint. Ezrin/Moesin Total 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.
