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

The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total Moesin assay is a sandwich immunoassay for quantitative detection of total 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 Moesin assay is a sandwich immunoassay for quantitative detection of total Moesin in cellular lysates using Alpha Technology.

Product Variants
Unit Size: 100 assay points
Part #:
ALSU-TMOES-A-HV
List Price
USD 694.00
Unit Size: 500 assay points
Part #:
ALSU-TMOES-A500
List Price
USD 2,346.00
Unit Size: 10,000 assay points
Part #:
ALSU-TMOES-A10K
List Price
USD 14,270.00
Unit Size: 50,000 assay points
Part #:
ALSU-TMOES-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).
USD 2,346.00
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Overview

Moesin, a member of the ERM protein family, links the actin cytoskeleton to the plasma membrane, playing an essential role in maintaining cell structure and signaling. Activated by phosphorylation at Thr558, Moesin facilitates interactions with actin filaments and membrane proteins. Upregulation of Moesin is associated with cancers like breast and colorectal cancer, promoting tumor growth and metastasis. Moesin also modulates immune cell functions, contributing to autoimmune conditions.

The AlphaLISA SureFire Ultra Human and Mouse Total Moesin Detection Kit is a sandwich immunoassay for the quantitative detection of total 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
Moesin
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 Phospho (Thr558)/Total 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). Moesin Phospho (Thr558) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for Total and 400 cells for Phospho (lysates diluted 1:10)) 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, Calyculin A triggered a dose-dependent increase in the levels of Phospho Moesin (Thr558) while Total Moesin levels remained unchanged.

Validation of Phospho (Thr558)/Total Moesin in Calyculin a Treated Cells
Validation of Phospho (Thr558)/Total 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). Moesin Phospho (Thr558) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for Total and 1,000 cells for Phospho (lysates diluted 1:4)) 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, Staurosporine triggered a dose-dependent decrease in the levels of Phospho Moesin (Thr558) while no significant changes were observed for Total Moesin.

Decrease of Phospho Moesin (Thr558) levels in Staurosporine Treated Cells
Specificity of Moesin Total assay

Specificity of the Moesin Total assay was assessed using Ezrin, Radixin and Moesin recombinant human proteins.

Recombinant Ezrin (Abcam, ab132943), Radixin (RayBiotech, 230-30097) and Moesin (RayBiotech, 230-30095) proteins were prepared at 0.5 µg/mL in Lysis Buffer. Moesin total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein 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.

As expected, Moesin Total assay reacts only to Moesin recombinant protein. This result demonstrates the specificity of the Moesin Total assay as these 3 proteins share more than 70% sequence identity.

Selectivity of Moesin Total Assay
Specificity of Moesin Total assay

Specificity of Moesin Total protein levels were assessed in HeLa wild type (WT) and HeLa Moesin knockout (KO) cells. Both cell lines were grown to confluency in a T75 flask in medium at 37°C, 5% CO2. Cells were lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were diluted 1:2 in Lysis Buffer and evaluated for Total Moesin using the AlphaLISA SureFire Ultra kit.

For the detection step, 10 µL of cell lysate (approximately 10,000 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 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, Total Moesin was only detected in the WT HeLa cells but not in the HeLa Moesin KO cell line.

Knockout Validation of Moesin Total Assay
Versatility of Moesin Total assay in various cell lines

Human and mouse cell lysates were diluted with Lysis Buffer to represent approximately 10,000 cells/datapoint. 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.

Moesin Total protein expression is varied depending on cell type. Moesin protein is highly expressed in HeLa, THP-1, A549 and HUVEC cell lines, with low or no expression in MCF7, RT-4 and SH-SY5Y cell lines.

Versatility of Moesin Total assay in various cell lines

 

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