NLRP3 Total Biotin Free Assay Kit Human

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 TLR4 overexpressing cells were seeded at 100,000 cells/well in a 96 well plate and treated with 1 μg/mL LPS for the indicated time points. Cells were lysed with 100 μL Lysis Buffer and assayed separately for Total NLRP3 and Phospho NFkB (Ser536) using respective SureFire® Biotin Free kits. Equivalent to approximately 10,000 cells/datapoint (NLRP3) or 1,000 cells/datapoint (Phospho NFkB).

Data obtained with a 2-plate, 2-incubation protocol. THP-1 TLR4 overexpressing cells were seeded at 150,000 cells/well in a 96 well plate and left untreated or treated with LPS (500 ng/mL, 4h), Nigericin (10 μM, 30 minutes) or LPS + Nigericin (LPS 4h + Nig 30 minutes). After treatment, cells were spun down, supernatant harvested and cells were lysed with 100 μL Lysis Buffer. NLRP3 Total levels were evaluated in the cell lysate using the SureFire® Biotin Free assay, while the presence of IL-1b was evaluated in the collected supernatants. equivalent to approximately 15,000 cells/datapoint.

Data obtained from measurement of NLRP3 levels in Peripheral Blood Mononuclear cells (PBMC). PBMCs were isolated from healthy donors and cells were lysed at 6 x 106 cells/mL with Lysis Buffer. Lysates were serially diluted in Lysis Buffer and assayed for NLRP3 Total using the SureFire® Biotin Free assay.

Data obtained from measurement NLRP3 in various cell types lysed with Lysis Buffer. Equivalent to approximately 4,000 adherent or 10,000 suspension cells/datapoint.