ATF-3 Total Biotin Free 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. RPMI 8226 cells were seeded at 200K cells/well in a 96-well plate and treated with 1 μg/mL of LPS for indicated time points. Cells were spun down, lysed with Lysis Buffer and assayed for Total ATF-3 using the SureFire® Biotin Free kit. Equivalent to approximately 30,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. RPMI 8226 cells were seeded at 200K cells/well in a 96-well plate and treated with 5 μM of TLR7/8 agonist R848 for indicated time points. Cells were spun down, lysed with Lysis Buffer and assayed for Total ATF-3 using the SureFire® Biotin Free kit. Equivalent to approximately 40,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. RPMI 8226 cells were seeded at 200K cells/well in a 96-well plate and treated with increasing concentrations of LPS for 4 hours. After treatment cells were spun down and lysed with Lysis Buffer and assayed separately for Total ATF-3 and IRF5 using respective SureFire® Biotin Free kits. Equivalent to approximately 30,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. RAW264.7 cells were seeded at 60K cells/well in a 96-well plate
and incubated overnight. Cells were treated with increasing concentrations of LPS for 4 hours. Cells were lysed with Lysis Buffer and assayed separately for Total ATF-3 and ERK using respective SureFire® Biotin Free kits. Equivalent to approximately 20,000 cells/datapoint for ATF-3 and 6,000 cells/datapoint for ERK.

HCT-116 wild type (WT) and HCT-116 ATF-3 knockout (Abcam ab266872) cells were cultured to confluency in T175 flask. Each flask was lysed with 4 mL of Lysis Buffer and lysate dilutions were assayed for ATF-3 using the SureFire® Biotin Free kit.

Data obtained from measurement of ATF-3 Total in various cell types lysed with Lysis Buffer. Equivalent to
approximately 20,000 cells/datapoint for adherent and 16,000 cells/datapoint for suspension cells.