AlphaLISA SureFire Ultra Human Total PKR Detection Kit, 500 Assay Points

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.

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.

Validation of PKR Total

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.

Versatility of Total PKR assay in various cell lines

Adherent cells were grown to confluency in a T175 flask at 37°C, 5% CO₂ and were lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at 1.6 x 10⁶ cells/mL.

Total PKR levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (5,000 adherent and 16,000 suspension cells) were 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.

Total PKR expression was detected in a wide range of human cell lines.

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.