STAT3 Total High Performance 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. THP-1 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium containing 100 nM PMA and incubated for 24 hours. The THP-1 derived macrophages were starved in HBSS + 0.1% BSA for 2 hours and then stimulated with increasing concentrations of IFNα for 20 minutes. After treatment, the cells were lysed with Lysis Buffer and Phospho (Tyr705) and Total STAT3 levels were evaluated using respective SureFire® Ultra™ kits. Equivalent to approximately 5,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. THP-1 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium containing 100 nM PMA and incubated for 24 hours. The THP-1 derived macrophages were starved in HBSS + 0.1% BSA for 2 hours and then stimulated with increasing concentrations of IL-6 for 20 minutes. After treatment, the cells were lysed with Lysis Buffer for 10 minutes and Phospho (Tyr705) and Total STAT3 levels were evaluated using respective SureFire® Ultra™ High Performance kits. Equivalent to approximately 5,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight. Cells were starved for 20 hours and then stimulated with increasing concentrations of IL-6 for 30 minutes. After treatment, the cells were lysed with Lysis Buffer and Phospho (Tyr705) and Total STAT3 levels were evaluated using respective SureFire® Ultra™ High Performance kits. Equivalent to approximately 4,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol RAW264.7 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight. Cells were starved for 2 hours and then stimulated with increasing concentrations of IFNγ for 20 minutes. After treatment, the cells were lysed with Lysis Buffer and Phospho (Tyr705) and Total STAT3 levels were evaluated using respective SureFire® Ultra™ High Performance kits. Equivalent to approximately 4,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. THP-1 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium containing 100 nM PMA and incubated for 24 hours. The THP-1 derived macrophages were treated with increasing concentrations of Ruxolitinib for 2 hours and then stimulated with 10 ng/mL IFNγ for 20 minutes. After treatment, the cells were lysed with Lysis Buffer for 10 minutes and Phospho (Tyr705) and Total STAT3 levels were evaluated using respective SureFire® Ultra™ kits. Equivalent to approximately 5,000 cells/datapoint.

Data obtained from measurement of STAT3 in various cell types lysed with Lysis Buffer. Equivalent to approximately 1,000 cells/datapoint (adherent cells) or 3,000 cells/datapoint (suspension cells).