SMAD3 is an intracellular signal transducer and transcription factor that mediates TGF-β and activin signaling to regulate cell growth, differentiation, and extracellular matrix production. Upon TGF-β receptor activation, SMAD3 is phosphorylated, enabling formation of complexes with SMAD4 that translocate to the nucleus. The SMAD3/SMAD4 complex regulates transcription of genes including collagens, fibronectin, and cyclin-dependent kinase inhibitors. SMAD3 plays context-dependent roles as both a tumor suppressor and tumor promoter. Loss of SMAD3 contributes to colorectal cancer, while excessive SMAD3 activation drives pathological fibrosis in multiple organs.
The AlphaLISA SureFire Ultra Human and Mouse High Specificity Phospho-SMAD3 (Ser423/425) is a sandwich immunoassay for the quantitative detection of phospho-SMAD3 (Ser423/425) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
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 enables cell 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 robust AlphaLISA SureFire Ultra quality.
Activation of SMAD3 Phospho (Ser423/425) in various cell models
HeLa and A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were starved for 1 hour and then treated with increasing concentrations of TGF-β for 90 minutes.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). SMAD3 Phospho (Ser423/425) and Total levels were evaluated using AlphaLISA SureFire Ultra High Specificity 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, TGF-β triggered a dose-dependent increase in the levels of SMAD3 Phospho (Ser423/425) while Total levels remain unchanged.
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.
