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AlphaLISA SureFire Ultra Human and Mouse Total Ezrin/Moesin Detection Kit, 500 Assay Points

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.

Product Variants
Unit Size: 100 assay points
Part #:
ALSU-TEM-A-HV
List Price
USD 694.00
Unit Size: 500 assay points
Part #:
ALSU-TEM-A500
List Price
USD 2,346.00
Unit Size: 10,000 assay points
Part #:
ALSU-TEM-A10K
List Price
USD 14,270.00
Unit Size: 50,000 assay points
Part #:
ALSU-TEM-A50K
List Price
USD 46,060.00
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
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Overview

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

Specifications

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

How it works

Total-AlphaLISA SureFire Ultra assay principle

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.

Assay Principle Total AlphaLISA SureFire Ultra

 

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 permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

2plates-assay-protocol-AlphaLISA-Surefire-Ultra.jpg
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 AlphaLISA SureFire Ultra quality.

1 plate assay protocol AlphaLISA Surefire Ultra

Assay validation

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.

HeLa cells treated with Calyculin
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.

A549 cells treated with Staurosporine
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.

Assay cross reactivity
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.

Evaluation of Ezrin/Moesin Total levels in various cell lines

Resources

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SDS, COAs, Manuals and more

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