NLRP3 Total Biotin Free Assay Kit 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. RAW 264.7 cells were seeded at 60K cells/well in a 96 well plate and incubated for 48 hours. Cells were treated with 500 ng/mL LPS for the indicated time points. Cells were lysed with Lysis Buffer and assayed separately for Total and Aggregate NLRP3 using respective SureFire® Biotin Free kits. Equivalent to approximately 1,200 cells/datapoint (Total) or 40,000 cells/datapoint (Aggregate).

Data obtained with a 2-plate, 2-incubation protocol. RAW 264.7 cells were seeded in T25 flasks and cultured to 90% confluency in complete medium. Cells were untreated or treated with LPS (500 ng/mL, 4h), Nigericin (10 μM, 1h) or LPS + Nigericin (LPS 4h + Nig 1h). Cells were lysed with Lysis Buffer and assayed separately for Total and Aggregate NLRP3 using respective SureFire® Biotin Free kits. Equivalent to approximately 5,000 cells/datapoint (Total) or 25,000 cells/datapoint (Aggregate).

Data obtained with a 2-plate, 2-incubation protocol. RAW 264.7 cells were seeded at 60K cells/well in a 96 well plate and incubated for 24 hours. Cells were treated with increasing concentrations of LPS for 4 hours. Cells were lysed with Lysis Buffer and assayed separately for Total and Aggregate NLRP3, and ERK Total using respective SureFire® Biotin Free kits. Equivalent to approximately 1,200 cells (Total NLRP3), 40,000 cells (Aggregate), or 12,000 cells/datapoint (Total ERK).

Data obtained from measurement NLRP3 in various cell types lysed with Lysis Buffer. Equivalent to approximately 4,000 adherent or 40,000 suspension cells/datapoint.