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AlphaLISA SureFire Ultra Human Total EGF Receptor E746A-750 Deletion Detection Kit, 10,000 Assay Points

The AlphaLISA™ SureFire® Ultra™ Human Total EGF Receptor E746A-750 Deletion assay is a sandwich immunoassay for quantitative detection of total EGF Receptor E746A-750 Deletion in cellular lysates using Alpha Technology.

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Feature	Specification
Application	細胞シグナル伝達
Protocol Time	2h at RT
Sample Volume	10 µL

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Product variants

Unit Size: 100 Assay Points

Part #:

ALSU-TEGFR-C-HV

Unit Size: 500 Assay Points

Part #:

ALSU-TEGFR-C500

Unit Size: 10,000 Assay Points

Part #:

ALSU-TEGFR-C10K

Unit Size: 50,000 Assay Points

Part #:

ALSU-TEGFR-C50K

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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Product information

Overview

The Epidermal Growth Factor Receptor (EGFR) is a receptor tyrosine kinase that binds EGF and related ligands to regulate cell proliferation, survival, differentiation, and migration. Upon ligand binding, EGFR undergoes dimerization and autophosphorylation, leading to activation of downstream signaling pathways including RAS/MAPK, PI3K/AKT, and PLCγ. EGFR plays a critical role in normal epithelial development and tissue homeostasis.

The E746_A750 deletion, one of the most prevalent EGFR exon 19 activating mutations, removes key amino acids within the kinase domain, destabilizing the inactive conformation of the receptor. This structural alteration results in ligand‑independent, constitutive EGFR activation and persistent downstream signaling that drives oncogenic transformation.

EGFR exon 19 deletions are particularly common in non‑small cell lung cancer (NSCLC) and are strongly associated with tumor growth, survival, and disease progression. These mutations represent well‑validated predictive biomarkers for response to EGFR‑targeted therapies. Small‑molecule tyrosine kinase inhibitors such as erlotinib, gefitinib, and osimertinib selectively inhibit mutant EGFR signaling and have become standard‑of‑care treatments for patients with EGFR exon 19‑deleted tumors, while monoclonal antibodies such as cetuximab and panitumumab are used in EGFR‑overexpressing cancers.

The AlphaLISA SureFire Ultra Human Total EGF Receptor E746A-750 Deletion Detection Kit is a sandwich immunoassay for the quantitative detection of total EGF receptor E746A-750 deletion 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

How it works

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.

2 plates assay protocol AlphaLISA Surefire Ultra Total assay

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.

1-plate-assay-protocol-AlphaLISA-Surefire-Ultra-Total-assay

Assay specificity/selectivity

EGF Receptor E746-A750 expression in various cell lines

The presence of the EGF Receptor E746-A750 deletion mutation was evaluated in various EGFR expressing cell lines using the AlphaLISA SureFire Ultra assay kit. Cells were grown to confluency in a T175 flask and lysed with Lysis Buffer at a density of 150,000 cells/mL. For the detection step, 10 µL of lysate (approximately 15,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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, EGF Receptor E746-A750 deletion mutation was only present in HCC-827 cells.

Specificity validation EGF Receptor E746-A750 deletion assay

Assay sensitivity

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HCC827 seeded in T175 flasks and cultured to confluence. Cells were lysed in 8 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysates were serially diluted in Lysis Buffer and assayed for EGF Receptor E746-A750 deletion levels using the AlphaLISA SureFire Ultra kit. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.

Approximate number of cells/datapoint is indicated. The dotted line represents assay background. The assay can detect Total EGF Receptor E746-A750 deletion expression down to 150 cells.

Sensitivity of EGF Receptor E746-A750 deletion assay

Specifications

Other specifications

Application	Cell Signaling
Automation Compatible	Yes
Brand	AlphaLISA SureFire Ultra
Detection Modality	Alpha
Product Group	Kit
Protocol Time	2h at RT
Sample Volume	10 µL
Shipping Conditions	Shipped in Blue Ice
Target	EGF Receptor
Target Class	Phosphoproteins
Target Species	Human
Technology	Alpha
Therapeutic Area	Oncology
Unit Size	10,000 Assay Points