AlphaLISA SureFire Ultra Human & Mouse Total IRF5 Detection Kit, 100 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 permits the cells' 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 AlphaLISA SureFire Ultra quality.

Validation of Phospho (Ser446)/Total IRF5 in Calyculin A treated cells

THP-1 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of Calyculin A for 2.5 hours.

After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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.

As expected, Calyculin A triggered a dose-dependent increase in the levels IRF5 Phospho (Ser446) while Total IRF5 levels remained unchanged.

Specificity of IRF5 Total assay

Total IRF5 protein levels were assessed in A549 wild type (WT) and A549 IRF5 knockout (KO) (Abcam, ab301006) cells. Both cell lines were grown to confluency in a T75 flask in medium at 37°C, 5% CO2. Each flask was lysed in 2 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were then evaluated for Total IRF5 using the AlphaLISA SureFire Ultra kit.

For the detection step, 10 µL of cell lysate 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 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, Total IRF5 protein was detected only in A549 WT cells and no signal was detected in the KO IRF5 A549 cell line, demonstrating assay specificity.

Evaluation of IRF5 Total expression in Peripheral Blood Mononuclear Cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors using Ficoll® Plaque Plus (Merck GE17-1440-02).

Cells were seeded in 96-well plate (400,000 cells/well) and lysed in 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Generated lysate was then serially diluted in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra Total IRF5. For the detection step, 10 µL of cell lysate (starting from approximately 40,000 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. The number of cells/datapoint is indicated on graph.

Differential expression of IRF5 Total in various cell lines

Human and mouse cell lysates were diluted with Lysis Buffer. Approximate number of cells/datapoint is indicated on graph. Total IRF5 levels were evaluated using the AlphaLISA SureFira Ultra assay kit. For the detection step, 10 µL of cell lysate 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.

Total IRF5 expression varies depending upon cell type. As expected, a very high level of expression was detected in human cell lines: THP-1, KARPAS-299 and U937 and mouse cell lines: RAW 264.7 and L929.