SMAD3 Phospho-(Ser423/425) HS Assay Kit Human and Mouse

The AlphaLISA™ SureFire® Ultra™ assay enables the rapid and sensitive detection of total and phosphorylated cellular proteins. AlphaLISA™ assays utilize two bead types: Acceptor Beads and Donor Beads. The Acceptor Bead is coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody which is labeled with a CaptSure™ tag. The Donor Bead is coated with streptavidin to capture the biotinylated antibody.  

In the presence of a target protein, the two target-specific antibodies bring Donor and Acceptor Beads into close proximity. When the Donor Beads are activated by a laser (680 nm), singlet oxygen is transferred to the Acceptor Bead leading to the production of an Alpha signal. The amount of light emission (615 nm) from the Acceptor Bead is directly proportional to the amount of target protein present in the sample. If an Acceptor Bead is not in close proximity (i.e. 200 nm) of a Donor bead, little to no signal is produced over background. 

The assay can be executed in a 1-plate or 2-plate assay protocol (Refer to Manual for more details).  

1-plate assay protocol: culturing of cells, treatment, lysis and assay are performed in a single well, enabling miniaturization in high throughput screening programs. 

2-plate assay protocol: cells are cultured and treated in a 96-well culture plate and lysates are then transferred into a separate plate for assay. This format allows the evaluation of multiple targets from a single lysate. 

Data obtained with a 2-plate, 2-incubation protocol. A549 cells were seeded at 40K cells/well in a 96-well plate and incubated overnight. Cells were starved for 1 hour and then treated with increasing concentrations of TGF-β for 90 minutes. Cells were lysed with Lysis Buffer and assayed separately for Phospho (Ser423/425) and Total SMAD3 using the SureFire® Ultra™ High Specificity kits. Equivalent to approximately 2,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. HeLa cells were seeded at 40K cells/well in a 96-well plate and incubated overnight. Cells were treated with increasing concentrations of TGF-β for 90 minutes. Cells were lysed with Lysis Buffer and assayed separately for Phospho (Ser423/425) and Total SMAD3 using the SureFire® Ultra™ High Specificity kits. Equivalent to approximately 2,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. A549 wild type (WT) and SMAD3 knockout (KO, Abcam ab263915) cells were grown to confluency in T175 flasks, starved for 1 hour, and stimulated with 50 ng/mL TGF-β for 90 minutes. Cells were lysed with Lysis Buffer and assayed for Phospho (Ser423/425) SMAD3 using the SureFire® Ultra™ High Specificity kit.