AlphaLISA Surefire Ultra Human Phospho-PKR (Thr451) Detection Kit, 100 Assay Points

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 permits the cells 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 AlphaLISA SureFire Ultra quality.

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.