Protein kinase R (PKR) is a kinase encoded by the EIF2AK2 gene and is involved in central cellular processes such as mRNA translation, transcriptional control, regulation of apoptosis, and proliferation. PKR plays a key role in the innate immunity pathway and is a mediator of the antiviral effects exerted by interferons. PKR also regulates eIF2α through phosphorylation. Dysregulation of PKR has been implicated in cancer, neurodegenerative disease, inflammation, and metabolic disorders.
The AlphaLISA SureFire Ultra Human Phospho-PKR (Thr451) assay is a sandwich immunoassay for the quantitative detection of phospho-PKR (Thr451) 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
Alpha SureFire kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
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.
PKR phosphorylation mediated by interferons
THP-1 cells were seeded in a 12-well plate (250,000 cells/well) in medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO2. THP-1 derived macrophages were then treated with 250 ng/mL of IFNα, IFNβ or IFNγ for a further 24 hours.
After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho PKR (Thr446 and Thr451) and Phospho eIF2α (Ser51) levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 25,000 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 IFNs induced phosphorylation of PKR (Thr446, Th451), leading to downstream phosphorylation of eIF2α (Ser51).
Activation of Phospho PKR (Thr451) in HeLa cells
HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were pretreated with 10 ng/mL IFNγ for 24 hours, then treated with increasing concentrations of Calyculin A for 30 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PKR Phospho (Thr451) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, pre-stimulation with IFNγ and subsequent treatment with Calyculin A increased phosphorylation of PKR (Thr451) with a 100-fold induction, with no significant changes to Total PKR levels.
Specificity of Phospho PKR (Thr451) assay
Specificity of the Phospho PKR (Thr451) assay was assessed by peptide competition.
Phospho (Thr451) and non-phospho PKR peptides were titrated into a fixed concentration of a positive control lysate (AN3 CA cells treated with Calyculin A for 4 hours).
Each peptide was then assessed for its ability to block antibody binding to the PKR Phospho site Thr451 using the Phospho (Thr451) PKR AlphaLISA SureFire Ultra assay kit. For the detection step, 10 µL of prepared 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.
PKR Phospho (Thr451) assay signal was blocked only by the Phospho Thr451 peptide, with no change in signal with Non-Phospho peptide, demonstrating assay specificity.
Assay sensitivity - cell lysate dilution
Cell lysate was prepared from AN3 CA cells cultured to confluency in T175 flasks at 37°C, 5% CO2. Cells were treated with 20 ng/mL of Calyculin A for 4 hours and then lysed in 5 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and Total PKR and Phospho (Thr451) levels were evaluated by AlphaLISA SureFire Ultra. 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 per datapoint is indicated. The dotted line represents assay background. The assay can detect PKR levels down to 1,000 cells/datapoint.
