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The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total Ezrin/Moesin assay is a sandwich immunoassay for quantitative detection of total Ezrin/Moesin in cellular lysates using Alpha Technology.
Feature | Specification |
---|---|
Application | Cell Signaling |
Protocol Time | 2h at RT |
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total Ezrin/Moesin assay is a sandwich immunoassay for quantitative detection of total Ezrin/Moesin in cellular lysates using Alpha Technology.
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:
AlphaLISA SureFire Ultra kits are compatible with:
AlphaLISA SureFire Ultra kits can be used for:
Application |
Cell Signaling
|
---|---|
Automation Compatible |
Yes
|
Brand |
AlphaLISA SureFire Ultra
|
Detection Modality |
Alpha
|
Protocol Time |
2h at RT
|
Shipping Conditions |
Shipped in Blue Ice
|
Target |
EM
|
Target Class |
Phosphoproteins
|
Target Species |
Human
Mouse
|
Technology |
Alpha
|
Therapeutic Area |
Autoimmunity
Oncology
|
Unit Size |
500 assay points
|
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted 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 targeted 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 the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
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 permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
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 AlphaLISA SureFire Ultra quality.
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.
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.
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.
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.
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