Inositol-Requiring Enzyme 1 Alpha (IRE1A) is an endoplasmic reticulum (ER) transmembrane protein that functions as both a kinase and endoribonuclease in the unfolded protein response (UPR). Upon ER stress, IRE1A undergoes oligomerization and autophosphorylation, activating its RNase domain to splice XBP1 mRNA, generating the active transcription factor XBP1s. XBP1s promotes expression of ER chaperones, lipid synthesis enzymes, and ER-associated degradation components to restore ER homeostasis. IRE1A also degrades ER-localized mRNAs through regulated IRE1-dependent decay (RIDD) to reduce ER protein load. Chronic IRE1A activation contributes to inflammation, apoptosis, and disease pathogenesis in cancer, diabetes, and neurodegenerative disorders. Modulating IRE1A activity represents a therapeutic opportunity for ER stress-related diseases.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-IRE1α (Ser724) is a sandwich immunoassay for the quantitative detection of phospho-IRE1α (Ser724) 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
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope 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 phosphorylated 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 phosphoprotein present in the sample.
Phospho-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 Phospho-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.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated 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.
Phosphorylation of IRE1 in tunicamycin treated cells
L929 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 tunicamycin for 4 hours.
After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRE1 Phospho (Ser724) and Total levels were evaluated using respective AlphaLISA SureFire Ultra 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.
Tunicamycin treatment triggered a significant increase in the levels of IRE1 Phospho (Ser724) and a modest increase in Total IRE1 levels.
Thapsigargin-mediated IRE1 phosphorylation
BeWo 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 Thapsigargin for 4 hours.
After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRE1 Phospho (Ser724) and Total levels were evaluated using respective AlphaLISA SureFire Ultra 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.
Thapsigargin treatment triggered a dose-dependent increase in the levels of Phospho (Ser724) IRE1 while total levels remained unchanged.
RPMI 8226 cells were seeded at 400,000 cells/well in a 96-well plate and treated with increasing concentrations of thapsigargin for 4 hours.
After treatment, the cells were spun down, washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRE1 Phospho (Ser724) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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.
Thapsigargin treatment resulted in a dose-dependent increase in the levels of Phospho (S724) IRE1 with no significant changes to the total levels.
