Revvity Sites Globally

Select your location.

*e-commerce not available for this region.

Australia

Austria

Belgium

Brazil *

Canada

China *

Denmark

Finland

France

Germany

Hong Kong (China) *

India *

Ireland

Italy

Japan *

Luxembourg

Mexico *

Netherlands

Norway

Philippines *

Republic of Korea *

Singapore *

Spain

Sweden

Switzerland

Thailand *

United Kingdom

United States

AlphaLISA SureFire Biotin-Free Mouse Total NLRP3 Detection Kit, 500 Assay Points

The AlphaLISA™ SureFire® Biotin-Free Mouse Total NLRP3 assay is a sandwich immunoassay for quantitative detection of total NLRP3 in cellular lysates using Alpha Technology.

View product information

Feature	Specification
Application	細胞シグナル伝達
Protocol Time	2h at RT
Sample Volume	10 µL

The AlphaLISA™ SureFire® Biotin-Free Mouse Total NLRP3 assay is a sandwich immunoassay for quantitative detection of total NLRP3 in cellular lysates using Alpha Technology.

View product information

Product variants

Compare all

Remove all

Add to compare Add to compare

削除

Unit Size: 100 Assay Points

Part #:

ASBF-TNLRP3-B-HV

Add to compare Add to compare

削除

Unit Size: 500 Assay Points

Part #:

ASBF-TNLRP3-B500

Add to compare Add to compare

削除

Unit Size: 10,000 Assay Points

Part #:

ASBF-TNLRP3-B10K

Add to compare Add to compare

削除

Unit Size: 50,000 Assay Points

Part #:

ASBF-TNLRP3-B50K

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

Request more information

Product information

Overview

NLRP3 (NOD-like Receptor Pyrin Domain-Containing 3) is a cytosolic pattern recognition receptor and the most extensively studied member of the NLR family that forms the core component of the NLRP3 inflammasome, a multiprotein innate immune signaling complex. NLRP3 is activated by a broad range of danger signals including ATP, uric acid crystals, cholesterol crystals, silica, asbestos, and mitochondrial reactive oxygen species, following a two-signal model in which an initial priming signal (typically NF-κB activation) upregulates NLRP3 expression and a second activation signal triggers inflammasome assembly. Upon activation, NLRP3 oligomerizes and recruits the adaptor protein ASC and pro-caspase-1 to form the inflammasome complex, leading to caspase-1 activation, proteolytic processing of pro-IL-1β and pro-IL-18 into their mature secreted forms, and induction of pyroptotic cell death through gasdermin D cleavage. NLRP3 inflammasome activation drives pathological inflammation in a wide range of diseases including gout, atherosclerosis, type 2 diabetes, Alzheimer's disease, Parkinson's disease, and inflammatory bowel disease. Selective NLRP3 inhibitors such as MCC950 (CMPD-4) and clinical-stage compounds including inzomelid and selnoflast are under active investigation as anti-inflammatory therapeutics across multiple indications.

The AlphaLISA SureFire Biotin-Free Mouse Total NLRP3 Detection Kit is a sandwich immunoassay for the quantitative detection of total NLRP3 in cellular lysates, using Alpha Technology.

Formats:

The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.

AlphaLISA SureFire Biotin Free kits are compatible with:

Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples

AlphaLISA SureFire Biotin Free kits can be used for:

Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies

How it works

Total-AlphaLISA SureFire Biotin Free assay principle

The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).

The Total-AlphaLISA SureFire Biotin Free assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with the CaptSure3 tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.

Total-AlphaLISA SureFire Ultra Biotin Free assay principle

Total-AlphaLISA SureFire Biotin Free two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

2 plates assay protocol alphalisa surefire ultra biotin free total assay

Total-AlphaLISA SureFire Biotin Free one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Biotin Free reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire quality.

1 plate assay protocol alphalisa surefire ultra biotin free total assay

Assay validation

LPS induces NLRP3 expression and aggregation in RAW 264.7 cells

RAW 264.7 cells were seeded in a 96 well plate (60,000 cells/well) in complete medium and incubated for 48 hours at 37°C, 5% CO₂.Cells were treated with 500 ng/mL of LPS for the indicated time points.

After treatment, the cells were lysed with Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total and Aggregate NLRP3 levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 1,200 cells (Total), 40,000 cells (Aggregate)) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

LPS treatment induced NLRP3 expression and aggregation within 1 hour, peaking at 4 hours of treatment.

Induction of Aggregate NLRP3 following LPS and Nigericin treatment

RAW 264.7 cells were seeded in T25 cell culture flasks and cultured to 90% confluency in complete medium at 37°C, 5% CO₂.Cells were left untreated or treated with LPS (500 ng/mL, 4h), Nigericin (10 µM, 1h) or LPS + Nigericin (LPS 4h + Nig 1h).

After treatment, each flask was lysed with 3 mL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total and Aggregate NLRP3 levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 5,000 cells (Total) or 25,000 cells (Aggregate) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

LPS induced a significant increase in Total and Aggregate NLRP3 levels, while treatment with LPS + Nigericin triggered a further 2-fold increase in Aggregate NLRP3 levels.

LPS induces NLRP3 expression and aggregation in a dose-dependent manner

RAW 264.7 cells were seeded in a 96 well plate (60,000 cells/well) in complete medium and incubated for 24 hours at 37°C, 5% CO₂.Cells were treated with increasing concentrations of LPS for 4 hours.

After treatment, the cells were lysed with Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total and Aggregate NLRP3, and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 1,200 cells (Total), 40,000 cells (Aggregate), or 12,000 cells (ERK)) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

LPS triggered a dose-dependent increase of Total and Aggregate NLRP3 levels while ERK Total levels remained unchanged.

Assay specificity/selectivity

Specific expression of NLRP3 Total (Mouse) in RAW 264.7 cells

Adherent cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂.Suspension cells were harvested, washed, and seeded in a 96-well plate (400,000 cells/well) in HBSS.

Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NLRP3 Total levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of cell lysate (4,000 adherent cells or 40,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, NLRP3 expression levels were only detected in RAW 264.7 cells.

Assay selectivity – Differential cell expression

Assay sensitivity

Assay Sensitivity - cell lysate dilution

Cell lysate was prepared from RAW 264.7 cells seeded in T175 flasks and cultured to confluence at 37°C, 5% CO₂.Cells were then treated with 500 ng/mL LPS for 4 hours and lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysates were serially diluted in Lysis Buffer and NLRP3 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells/datapoint is indicated on the graph. The assay can detect NLRP3 down to approximately 100 cells/datapoint. Assay background is represented with a dotted line.

Assay sensitivity – cell lysate dilution

Specifications

Other specifications

Application	Cell Signaling
Automation Compatible	Yes
Brand	AlphaLISA SureFire Biotin-Free
Detection Modality	Alpha
Product Group	Kit
Protocol Time	2h at RT
Sample Volume	10 µL
Shipping Conditions	Shipped in Blue Ice
Target	NLRP3
Target Class	Phosphoproteins
Target Species	Mouse
Technology	Alpha
Therapeutic Area	Autoimmunity Inflammation
Unit Size	500 Assay Points