NLRP6 Antibody, Biotin conjugated

What is NLRP6 and what are its primary cellular functions?

NLRP6 (NOD-like receptor family pyrin domain containing 6) is a 99 kDa protein belonging to the NLR family that functions as a critical sensor component of the NLRP6 inflammasome. This multifunctional protein plays essential roles in several biological processes, including inflammasome formation, intestinal homeostasis, and antiviral immunity. NLRP6 mediates inflammasome activation in response to various pathogen-associated signals, resulting in the maturation and secretion of pro-inflammatory cytokines IL-1β and IL-18 . Its cellular localization spans the cytoplasm, nucleus, and cell membrane, enabling diverse molecular interactions .

NLRP6 recognizes specific pathogen-associated molecular patterns (PAMPs) including lipoteichoic acid (LTA) from Gram-positive bacteria and double-stranded RNA (dsRNA) from viruses . Within the intestinal environment, NLRP6 maintains epithelial integrity and modulates inflammatory responses to prevent colonization by harmful bacteria such as Prevotella species, thereby protecting against colitis development . Additionally, NLRP6 may contribute to arginine-vasopressin (AVP)-mediated regulation of renal salt-water balance, glucose and lipid metabolism, apoptosis, and cell cycle progression .

How does NLRP6 contribute to antiviral immunity?

NLRP6 plays a crucial role in antiviral immunity through both inflammasome-dependent and inflammasome-independent mechanisms. Research has revealed that NLRP6 participates in the recognition of viral RNA in coordination with the RNA helicase Dhx15 . This interaction is particularly important for controlling enteric virus infections such as EMCV (encephalomyocarditis virus).

The mechanism involves a multiprotein complex where NLRP6 preferentially binds to dsRNA analogs like poly I:C . Following viral infection, NLRP6 enhances the interaction between Dhx15 and MAVS (mitochondrial antiviral signaling protein), forming the Dhx15-Nlrp6-MAVS signaling axis that restricts viral replication . Experimental evidence demonstrates that Nlrp6-deficient cells and animals exhibit impaired expression of type I/III interferon-stimulated genes (ISGs) and increased viral loads compared to wild-type counterparts . Importantly, this antiviral function occurs independently of caspase-1 activation, distinguishing it from the canonical inflammasome pathway typically associated with NLRP6 .

What are the key applications for NLRP6 antibody, biotin conjugated?

The biotin-conjugated NLRP6 antibody offers researchers versatility across multiple experimental applications. Based on validated testing, this reagent is suitable for:

• Enzyme-Linked Immunosorbent Assay (ELISA): Enables quantitative detection of NLRP6 in solution with recommended dilutions of 1:500-1000 .

• Immunohistochemistry (IHC):

  • Paraffin-embedded sections (IHC-P): Allows visualization of NLRP6 expression patterns in fixed tissue sections at dilutions of 1:200-400 .

  • Frozen sections (IHC-F): Provides detection in frozen tissue preparations at dilutions of 1:100-500 .

• Immunoprecipitation (IP): Facilitates isolation and purification of NLRP6 protein complexes using 1-2 μg of antibody per reaction .

The biotin conjugation specifically enhances detection sensitivity through avidin/streptavidin amplification systems, making this antibody particularly valuable for examining tissues with low NLRP6 expression levels. Additionally, the antibody demonstrates confirmed reactivity with mouse and rat samples, with predicted cross-reactivity to human samples .

What sample types and tissue sources are optimal for NLRP6 detection?

When designing experiments utilizing biotin-conjugated NLRP6 antibody, researchers should consider the following validated sample types:

For immunohistochemical applications in human samples, optimal results require careful attention to antigen retrieval methods. The recommended approach involves using TE buffer at pH 9.0, though citrate buffer at pH 6.0 represents an alternative method for certain sample types . When working with intestinal tissues, researchers should note that NLRP6 expression is particularly enriched in intestinal epithelial cells, where it performs critical functions in maintaining epithelial integrity and regulating host-microbiota interactions .

How should researchers optimize protocols for NLRP6 detection using biotin-conjugated antibodies?

Optimizing experimental protocols for biotin-conjugated NLRP6 antibody requires careful attention to several key parameters:

• Antigen Retrieval Methods:

  • For formalin-fixed paraffin-embedded tissues, use TE buffer at pH 9.0 as the primary method, with citrate buffer at pH 6.0 as an alternative .

  • Complete retrieval time should be optimized based on tissue type and fixation duration.

• Blocking Considerations:

  • Implement an additional avidin/biotin blocking step to minimize non-specific binding, particularly important when working with tissues containing endogenous biotin.

  • The standard blocking solution should contain TBS (0.01M, pH 7.4) with 1% BSA .

• Signal Development Systems:

  • For optimal sensitivity, employ streptavidin-HRP or streptavidin-fluorophore conjugates depending on the desired detection method.

  • When using chromogenic detection, DAB (3,3'-diaminobenzidine) provides strong contrast for visualizing NLRP6 localization.

• Titration Requirements:

  • Each new tissue type or experimental system requires antibody titration to identify optimal working concentrations.

  • Begin with the manufacturer's recommended dilution ranges (1:50-1:500 for IHC applications) and adjust based on signal-to-noise ratio .

What controls should be implemented to validate NLRP6 antibody specificity?

Rigorous validation of antibody specificity is essential for generating reliable data with biotin-conjugated NLRP6 antibody. Researchers should implement the following control strategies:

• Positive Controls:

  • Mouse or rat small intestine tissue samples serve as ideal positive controls due to their high endogenous NLRP6 expression .

  • For cell-based assays, A549 cells have been validated for NLRP6 detection .

• Negative Controls:

  • Include isotype-matched, biotin-conjugated rabbit IgG at equivalent concentrations to assess non-specific binding.

  • Where available, tissues or cells from NLRP6 knockout models provide definitive negative controls.

• Peptide Competition Assays:

  • Pre-incubate the antibody with excess immunizing peptide (from the 351-450/892 region of human NLRP6) to demonstrate binding specificity .

  • Signal abolishment confirms antibody specificity for the target epitope.

• Molecular Weight Verification:

  • When performing western blot analysis in conjunction with other applications, verify detection at the correct molecular weight range (98-105 kDa) .

How can NLRP6 biotin-conjugated antibodies be used to study inflammasome formation?

The study of NLRP6 inflammasome formation represents a complex area of research where biotin-conjugated antibodies offer significant advantages. Recent findings have revealed that NLRP6 undergoes liquid-liquid phase separation (LLPS) following ligand binding, a critical step in inflammasome assembly . Researchers can leverage biotin-conjugated NLRP6 antibodies to investigate this process through several approaches:

• Co-immunoprecipitation Studies:

  • Use the biotin-conjugated antibody with streptavidin beads to isolate NLRP6-containing protein complexes.

  • Analyze precipitated complexes for interaction partners such as ASC and caspase-1/11, which are essential components of the inflammasome .

  • This approach can be particularly effective for identifying differential complex formation following stimulation with LTA or dsRNA.

• Proximity Ligation Assays (PLA):

  • Combine the biotin-conjugated NLRP6 antibody with antibodies against other inflammasome components.

  • Visualize molecular proximity (<40 nm) between NLRP6 and interacting partners such as ASC during inflammasome assembly.

  • This technique enables spatial and temporal analysis of inflammasome formation in intact cells.

• Immunofluorescence Microscopy of Phase Separation:

  • Utilize streptavidin-fluorophore conjugates to visualize NLRP6 condensates forming during LLPS.

  • Implement time-course experiments to monitor the kinetics of NLRP6 redistribution following pathogen recognition.

  • Combine with super-resolution microscopy techniques to resolve fine structural details of inflammasome architecture.

What approaches can be used to investigate NLRP6's interaction with viral RNA?

The discovery that NLRP6 binds viral RNA and contributes to antiviral immunity independently of inflammasome activation opens new research avenues . Biotin-conjugated NLRP6 antibody facilitates several methodological approaches to explore these interactions:

• RNA-Protein Complex Immunoprecipitation:

  • Utilize the biotin-conjugated antibody to precipitate NLRP6-RNA complexes from virus-infected cells.

  • Analyze co-precipitated RNA through RT-PCR or RNA sequencing to identify viral RNA sequences preferentially bound by NLRP6.

  • Research has demonstrated that NLRP6 preferentially binds long dsRNA analogues such as poly I:C over other RNA structures .

• Analysis of Dhx15-Dependent RNA Binding:

  • Implement siRNA knockdown of Dhx15 followed by immunoprecipitation with biotin-conjugated NLRP6 antibody.

  • Quantify co-precipitated viral RNA to assess the dependency of NLRP6-RNA interactions on Dhx15 helicase activity.

  • Previous research demonstrated significantly reduced NLRP6-viral RNA binding in Dhx15-depleted cells .

• Visualization of NLRP6-RNA Interactions:

  • Combine fluorescently-labeled viral RNA or poly I:C with streptavidin detection of biotin-conjugated NLRP6 antibody.

  • Employ confocal microscopy to track co-localization during infection or stimulation.

  • This approach can reveal spatial dynamics of NLRP6 redistribution in response to viral infection.

How can researchers utilize NLRP6 antibodies to explore its inflammasome-independent functions?

Beyond its role in inflammasome formation, NLRP6 exhibits several inflammasome-independent functions that can be investigated using biotin-conjugated antibodies:

• Analysis of MAVS-Dhx15-NLRP6 Signaling Axis:

  • Use immunoprecipitation with biotin-conjugated NLRP6 antibody followed by western blotting for MAVS and Dhx15.

  • Compare complex formation in wild-type versus caspase-1-deficient backgrounds to delineate inflammasome-independent pathways.

  • Research has shown that NLRP6 enhances MAVS-Dhx15 interactions following viral infection, which occurs independently of inflammasome activation .

• Interferon-Stimulated Gene (ISG) Expression Analysis:

  • Correlate NLRP6 expression patterns (detected via biotin-conjugated antibody) with ISG expression in response to viral infection.

  • Compare responses in caspase-1-deficient versus NLRP6-deficient systems to distinguish inflammasome-dependent and independent pathways.

  • Previous studies demonstrated that NLRP6-deficient cells and tissues show impaired ISG expression despite normal caspase-1 function .

• Vasopressin Receptor Function Studies:

  • Investigate NLRP6's reported function as an arginine-vasopressin V2 receptor using biotin-conjugated antibodies to track subcellular localization.

  • Analyze co-localization with canonical vasopressin receptors and downstream signaling components.

  • NLRP6 has been implicated in AVP-mediated regulation of renal salt-water balance, glucose metabolism, and cell cycle regulation .

What are common issues when using biotin-conjugated NLRP6 antibodies and how can they be resolved?

Researchers working with biotin-conjugated NLRP6 antibodies may encounter several technical challenges. The following table outlines common issues and their solutions:

What factors might affect the stability and performance of biotin-conjugated NLRP6 antibodies?

The stability and performance of biotin-conjugated NLRP6 antibodies depend on several key factors:

• Storage Conditions:

  • Maintain at -20°C for optimal stability, with manufacturer specifications indicating 12-month stability under these conditions .

  • The inclusion of 50% glycerol in the storage buffer prevents freeze-thaw damage during repeated use .

  • Aliquoting is recommended for antibodies without stabilizing additives to minimize freeze-thaw cycles.

• Buffer Composition:

  • The standard formulation contains 0.01M TBS (pH 7.4) with 1% BSA and 0.03% Proclin300 as a preservative .

  • Sodium azide (0.02%) may be included in some formulations as an antimicrobial agent .

  • These components help maintain antibody stability and prevent microbial contamination during storage.

• Conjugation Stability:

  • The biotin-antibody linkage can degrade over time, particularly under suboptimal storage conditions.

  • Periodic quality control testing using known positive samples is recommended to verify conjugate performance.

  • Signal reduction over time may indicate conjugation degradation rather than antibody loss.

• Working Solution Preparation:

  • Diluted working solutions have reduced stability compared to stock concentrations.

  • Prepare fresh dilutions for each experiment when possible.

  • If working solutions must be stored, maintain at 4°C for no more than 1 week with appropriate preservatives.

How might emerging research on NLRP6 influence antibody development and applications?

Recent discoveries about NLRP6 function suggest several promising directions for future antibody development and application:

• Targeting Functional Domains:

  • As research clarifies the specific domains involved in RNA binding, ligand recognition, and inflammasome assembly, domain-specific antibodies could become valuable tools.

  • Antibodies targeting the RNA-binding region could help elucidate the structural basis for NLRP6's preference for dsRNA.

  • The recent discovery of NLRP6's liquid-liquid phase separation (LLPS) capability suggests that antibodies recognizing LLPS-specific conformational epitopes might provide unique insights into inflammasome assembly dynamics .

• Investigating Tissue-Specific Functions:

  • While NLRP6's intestinal functions are well-studied, its roles in other tissues remain less clear.

  • Antibodies optimized for various tissue types could help map NLRP6 expression and function throughout the body.

  • The reported vasopressin receptor function suggests potential roles in renal and metabolic processes that merit further investigation .

• Therapeutic Potential:

  • As NLRP6's protective role against colitis becomes better understood, antibody-based approaches for monitoring or modulating its activity might have clinical applications.

  • Antibodies capable of distinguishing between active and inactive NLRP6 conformations could serve as biomarkers for intestinal inflammation.

What methodological advances could enhance NLRP6 antibody applications?

Several technological advances could significantly enhance the utility of NLRP6 antibodies in research applications:

• Multiplexed Detection Systems:

  • Development of multiplexed imaging approaches combining biotin-conjugated NLRP6 antibodies with other inflammasome component antibodies could provide comprehensive visualization of assembly dynamics.

  • Mass cytometry (CyTOF) applications using metal-conjugated NLRP6 antibodies would enable high-dimensional analysis of inflammasome components at the single-cell level.

• Live-Cell Imaging Adaptations:

  • Converting antibody-derived binding fragments into cell-permeable nanobodies could enable live-cell tracking of NLRP6 dynamics during inflammasome formation.

  • This approach would overcome limitations of fixed-cell imaging in capturing the temporal aspects of LLPS and inflammasome assembly.

• Antibody-Based Proximity Sensors:

  • Engineering split-fluorescent protein systems linked to NLRP6 antibody fragments could create molecular sensors that fluoresce only upon inflammasome assembly.

  • Such tools would provide quantitative readouts of NLRP6 activity in living systems.