AlphaLISA SureFire Ultra Human Total IRF2 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 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.

Knockout validation of IRF2 Total assay

Total IRF2 levels were assessed in A549 wild type (WT) and IRF2 KO (Abcam ab267015) cells cultured to confluence in T175 flasks at 37°C, 5% CO₂,and lysed in 3 mL of Lysis Buffer.

Lysates were serially diluted and IRF2 levels were evaluated using the AlphaLISA SureFire Ultra assay. 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.

IRF2 was detected in WT cells but not in KO cells, confirming the specificity of the assay for the detection of IRF2 protein. The dotted line represents assay background.

IRF2 expression in human 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 at 200,000 cells/mL.

IRF2 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, Total IRF2 protein is highly expressed in B lymphoma cell lines like RPMI 8226 and Karpas 299, with low levels of basal expression detected in Daudi and U937.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from Karpas 299 cells. Cells were harvested, washed with HBSS and lysed to a final density of 5 x 10⁶ cells/mL in Lysis Buffer.

Lysates were serially diluted in Lysis Buffer and IRF2 levels were evaluated using the AlphaLISA SureFire Ultra kit. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL 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 IRF2 expression in less than 1000 cells. Assay background is indicated by the dotted line.