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AlphaLISA SureFire Ultra Human and Mouse IRF6 Dimer Detection Kit, 100 Assay Points

The AlphaLISA™ SureFire® Ultra™ Human and Mouse IRF6 Dimer assay is a sandwich immunoassay for quantitative detection of the IRF6 dimer in cellular lysates using Alpha Technology.

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Feature	Specification
Application	Cell Signaling
Protocol Time	2h at RT
Sample Volume	30 µL

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The AlphaLISA™ SureFire® Ultra™ Human and Mouse IRF6 Dimer assay is a sandwich immunoassay for quantitative detection of the IRF6 dimer in cellular lysates using Alpha Technology.

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ALSU-AIRF6-A-HV

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USD 737.00

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ALSU-AIRF6-A500

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USD 2,490.00

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ALSU-AIRF6-A10K

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USD 14,982.00

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ALSU-AIRF6-A50K

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USD 47,624.00

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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).

Product information

Overview

Interferon Regulatory Factor 6 (IRF6) is a transcription factor of the IRF family with critical roles in epithelial development, keratinocyte differentiation, and craniofacial morphogenesis. Unlike many IRF family members primarily associated with immune signaling, IRF6 is most prominently expressed in epithelial tissues and is essential for normal palate, lip, and skin development. IRF6 drives expression of genes involved in keratinocyte terminal differentiation and suppresses epithelial proliferation by regulating cyclin D1 and other cell cycle regulators. Loss-of-function mutations in IRF6 are causative for Van der Woude syndrome and popliteal pterygium syndrome, two autosomal dominant disorders characterized by cleft lip and palate. In cancer, IRF6 has been identified as a tumor suppressor in oral, breast, and skin cancers, where reduced IRF6 activity correlates with increased proliferation and invasiveness.

IRF6 dimerization represents a key step in the activation and transcriptional function of this epithelial regulatory transcription factor. Like other IRF family members, IRF6 contains an N-terminal DNA-binding domain and a C-terminal IRF association domain (IAD) that mediates homo- and heterodimerization. Dimerization of IRF6 is required for high-affinity DNA binding at composite regulatory elements and for full transcriptional activation of target genes involved in keratinocyte differentiation and epithelial barrier formation. The IRF6 dimer interface has been shown to be disrupted by several disease-associated mutations found in Van der Woude and popliteal pterygium syndrome patients, underscoring the functional importance of the dimeric state. Detection of the IRF6 dimer, as distinct from total or phosphorylated IRF6 monomer, provides insight into the oligomeric activation state of IRF6 and its capacity for transcriptional engagement, which is particularly relevant in studies of epithelial development, wound healing, and oral cancer biology.

The AlphaLISA SureFire Ultra Human and Mouse IRF6 Dimer Detection Kit is a sandwich immunoassay for the quantitative detection of IRF6 dimer 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

Assay validation

PMA mediated IRF6 activation in various cell lines

HT-1376 and A431 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of PMA for 1 hour.

After treatment, the cells were lysed with 50 µL or 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Ser424) and Dimer IRF6 levels were evaluated using AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for IRF6 Phospho and 8,000 cells for IRF6 Dimer) 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, treatment with PMA induced IRF6 phosphorylation and subsequent dimerization with no change to Total ERK levels (data not shown).

Pharmacological validation (activation) of Phospho (Ser424) IRF6

Pharmacological validation (activation) of IRF6 dimer

PMA mediated IRF6 activation in mouse macrophages

RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with 50 nM of PMA for 1 hour.

After treatment, the cells were lysed with 50 µL or 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Ser424) and Dimer IRF6 levels were evaluated using AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for Phospho IRF6, 8,000 cells for Dimer) 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, treatment with PMA induced IRF6 phosphorylation and subsequent dimerization with no change to Total ERK levels (data not shown).

Assay sensitivity

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HT-1376 cells cultured to confluence in T175 flasks at 37°C, 5% CO₂. Cells were treated with 50 nM PMA for 1 hour and lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysates were serially diluted and Phospho (Ser424) and Dimer IRF6 levels were evaluated using AlphaLISA SureFire Ultra assays. 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 on the graph. The assay can detect IRF6 Dimer in less than 2,000 cells. The dotted line represents assay background.

Assay sensitivity of Phospho (Ser424) IRF6

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	30 µL
Shipping Conditions	Shipped in Blue Ice
Target	IRF6
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	Alpha
Therapeutic Area	Inflammation Oncology
Unit Size	100 Assay Points