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AlphaLISA SureFire Biotin-Free Human and Mouse Total ERK1/2 Detection Kit, 100 Assay Points

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AlphaLISA SureFire Biotin-Free Human and Mouse Total ERK1/2 Detection Kit, 100 Assay Points
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AlphaLISA SureFire Biotin Free Total Kit Schematic
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The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total ERK1/2 assay is a sandwich immunoassay for quantitative detection of total ERK1/2 in cellular lysates using Alpha Technology.

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Feature Specification
Application Cell Signaling
Protocol Time 2h at RT

The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total ERK1/2 assay is a sandwich immunoassay for quantitative detection of total ERK1/2 in cellular lysates using Alpha Technology.

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Product Variants
Unit Size: 100 assay points
Part #:
ASBF-TERK-A-HV
Unit Size: 500 assay points
Part #:
ASBF-TERK-A500
Unit Size: 10,000 assay points
Part #:
ASBF-TERK-A10K
Unit Size: 50,000 assay points
Part #:
ASBF-TERK-A50K
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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AlphaLISA-SureFire-Biotin-Free-Total-Primary
AlphaLISA-SureFire-Biotin-Free-Total-Primary
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AlphaLISA SureFire Biotin-Free Human and Mouse Total ERK1/2 Detection Kit, 100 Assay Points
AlphaLISA-SureFire-Biotin-Free-Total-Primary
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AlphaLISA-SureFire-Biotin-Free-Total-Primary
AlphaLISA SureFire Biotin Free Total Kit Schematic
AlphaLISA SureFire Biotin-Free Human and Mouse Total ERK1/2 Detection Kit, 100 Assay Points
AlphaLISA-SureFire-Biotin-Free-Total-Primary
AlphaLISA-SureFire-Biotin-Free-Total-Primary
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Product information

Overview

Extracellular signal-regulated kinases 1/2 (ERK1/2) are central components of the MAPK signaling cascade, regulating cell growth, survival, and differentiation. Activated by MEK1/2 through phosphorylation, ERK1/2 phosphorylate diverse cytoplasmic and nuclear targets. ERK1/2 dysregulation is associated with cancer, driving tumor proliferation and resistance to therapy, and with neurodegenerative diseases like Alzheimer's, where it influences amyloid plaque formation.

The AlphaLISA SureFire Biotin-Free Human and Mouse Total ERK1/2 Detection Kit is a sandwich immunoassay for the quantitative detection of total ERK1/2 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 Biotin Free kits are compatible with:

  • Cell and tissue lysates
  • Antibody modulators
  • Biotherapeutic antibodies
  • Biotin rich samples

AlphaLISA SureFire Biotin Free 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 Biotin-Free
Detection Modality
Alpha
Protocol Time
2h at RT
Shipping Conditions
Shipped in Blue Ice
Target
ERK1/2
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
Alpha
Therapeutic Area
Neuroscience
Oncology
Unit Size
100 assay points

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AlphaLISA SureFire Biotin-Free Human and Mouse Total ERK1/2 Detection Kit, 100 Assay Points
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AlphaLISA SureFire Biotin-Free Human and Mouse Total ERK1/2 Detection Kit, 100 Assay Points
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How it works

Total-AlphaLISA SureFire Biotin Free assay principle

The Total-AlphaLISA Surefire Biotin Free assay measures the expression level of a protein target in a cell lysate.

The Total-AlphaLISA Surefire 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 a proprietary CaptSure 3 agent to capture one of the detection antibodies, which is labeled with CaptSure 3 tag. 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.

Total-AlphaLISA SureFire Ultra Biotin Free assay principle

 

Total-AlphaLISA SureFire Biotin Free 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 Biotin Free 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.

2 plates assay protocol alphalisa surefire ultra biotin free total assay
Total-AlphaLISA SureFire one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Biotin Free 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 quality.

1 plate assay protocol AlphaLISA Surefire Ultra Total assay

Assay validation

Validation of Phospho (Thr202/Tyr204)/Total ERK1/2 in PMA treated cells

Jurkat cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of PMA for 15 minutes.

After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, PMA triggered a dose-dependent increase in the levels of Phospho ERK1/2 (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Pharmacological Validation of Total ERK1/2
Validation of Phospho (Thr202/Tyr204)/Total ERK1/2 in Anti-CD3 treated cells

Jurkat cells were seeded in a 96-well plate (200,000 cells/well) in serum-free RPMI 1640 medium. The cells were starved for 1 hour and then treated with increasing concentrations of Anti-CD3 antibody for 5 minutes. After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, Anti-CD3 triggered a dose-dependent increase in the levels of Phospho ERK1/2 (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Pharmacological Validation of Total ERK1/2
Validation of Phospho (Thr202/Tyr204)/Total ERK1/2 in Anti-IgM treated cells

Ramos cells were seeded in a 96-well plate (200,000 cells/well) in serum free RPMI 1640 medium. The cells were starved for 1 hour and then treated with increasing concentrations of Anti-IgM for 20 minutes. After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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.

Treatment with Anti-IgM triggered a dose-dependent increase in the levels of Phospho ERK1/2 (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Validation of Phospho (Thr202/Tyr204)/Total ERK1/2 in Anti-IgM Treated Cells

Validation of Phospho (Thr202/Tyr204)/Total ERK1/2 in AG1478 treated cells

PC3 cells were seeded in a 96-well plate (40,000 cells/well) in complete Ham’s F-12K (Kaighn’s) medium and incubated overnight at 37°C, 5% CO2. The cells were treated for 2 hours with increasing concentrations of AG1478 and then stimulated with 1 ng/mL of EGF for 30 minutes.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, AG1478 triggered a dose-dependent decrease in the levels of Phospho ERK (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Validation of Phospho (Thr202/Tyr204)/Total ERK1/2 in AG1478 Treated Cells

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