AlphaLISA SureFire Ultra Human and Mouse High Specificity Phospho-SMAD3 (Ser423/425) Detection Kit, 10,000 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.

Specificity of SMAD3 Phospho (Ser423/425) High Specificity Assay

SMAD3 Phospho (Ser423/425) levels were assessed in A549 wild type (WT) and SMAD3 KO (Abcam, ab263915) cell lines cultured to confluency in T175 flasks, starved for 1 hour, and stimulated with 50 ng/mL TGF-β for 90 minutes.

Each flask was lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and evaluated for SMAD3 Phospho (Ser423/425) using the AlphaLISA SureFire Ultra High Specificity assay. For the detection step, 10 µL of cell lysate (approximately 12,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL 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.

SMAD3 Phospho (Ser423/425) was only detected in treated WT cells, demonstrating assay specificity.

Assay sensitivity - cell lysate

Cell lysate was prepared from RAW 264.7 cells cultured to confluence in a T175 flask and stimulated with 50 ng/mL TGF-β for 90 minutes. Cells were lysed with 6 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and SMAD3 Phospho (Ser423/425) and Total levels were evaluated using AlphaLISA SureFire Ultra High Specificity assays. 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 SMAD3 Phospho (Ser423/425) down to 2,500 cells/datapoint.