AlphaLISA SureFire Ultra Human Total IRF1 Detection Kit, 50,000 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.

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium at 37°C, 5% CO₂. The cells were treated with increasing concentrations of IFNβ or IFNγ for 4 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). IRF1 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 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, treatment with IFNβ or IFNγ induced expression of IRF1 with no change to Total ERK levels.

A431 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium at 37°C, 5% CO₂. The cells were treated with increasing concentrations of IFNβ or IFNγ for 4 hours.

After treatment, cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF1 and ERK Total levels were evaluated using AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, treatment with IFNβ or IFNγ induced expression of IRF1 with no change to ERK Total levels.

Knockout validation of Total IRF1 assay

IRF1 levels were assessed in A549 wild type (WT) and IRF1 KO (Abcam, ab267042) cell lines cultured to confluency in T175 flasks at 37°C, 5% CO₂ and treated with 10 ng/mL of IFNβ for 4 hours.

Each flask was lysed with 6 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm) and IRF1 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of lysate (approximately 20,000 cells) 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.

IRF1 was detected in WT cells but not in KO cells, confirming the specificity of this assay for IRF1 protein.

IRF1 expression in various cell lines

Adherent cells were cultured to confluence in T175 flasks at 37°C, 5% CO₂ and lysed with Lysis Buffer at a density of 200,000 cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer to a final density of 200,000 cells/mL.

IRF1 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 20,000 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, basal IRF1 expression is higher in A431, Jurkat and RPMI 8226 cells, lower levels were observed in HeLa, A549 and U937 cells.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from THP-1 cells treated with 10 ng/mL of IFNγ for 4 hours at 1 x 10⁶ cells/mL. After treatment, cells were washed with HBSS and lysed at a final density of 2 x 10⁶ cells/mL.

Lysates were serially diluted and IRF1 levels were evaluated using 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/datapoint is indicated on the graph. The assay can detect IRF1 expression in less than 300 cells. Assay background is represented by the dotted line.