NLRP6 Antibody, HRP conjugated

What is NLRP6 and what cellular functions does it mediate?

NLRP6 (NOD-like receptor family pyrin domain containing 6) is a pattern recognition receptor that forms inflammasome complexes with ASC and caspase-1 or caspase-11. This complex plays a crucial role in cleaving pro-interleukin-1β (IL-1β) and IL-18 into their biologically active forms. NLRP6 is particularly important for the maintenance of epithelial integrity and has been implicated in various inflammatory conditions. NLRP6 is also known by other names including NACHT, LRR and PYD domains-containing protein 6, NALP6, and PYPAF5 . Recent research has demonstrated that NLRP6 can detect endolysosomal damage caused by both sterile triggers and bacterial pathogens such as Listeria monocytogenes, activating inflammatory responses in human cells .

What are the recommended dilutions for NLRP6 antibody across different applications?

The optimal dilution of NLRP6 antibody varies by application method:

These dilutions provide a starting point, but researchers should note that optimal concentration is sample-dependent. It is highly recommended to titrate the antibody in each testing system to obtain optimal results . For HRP-conjugated versions, similar dilution ranges typically apply, though specific product documentation should be consulted for confirmation.

What sample types have been validated for NLRP6 antibody detection?

NLRP6 antibodies have been validated in various sample types including:

These validations indicate the versatility of NLRP6 antibodies across multiple species (human, mouse, rat) and sample types . When using HRP-conjugated versions, these same sample types should be appropriate for detection.

How does NLRP6 activation mechanism differ from other inflammasome sensors like NLRP3?

• Unlike NLRP3, NLRP6 is not activated by nigericin or imiquimod, demonstrating distinct ligand specificity .

• NLRP6 activation by Listeria infection is not inhibited by extracellular potassium (which typically blocks potassium efflux-dependent NLRP3 activation) .

• The NACHT domain appears to be the critical sensor domain in NLRP6, with chimeric studies showing that transplanting the NLRP6 NACHT domain into NLRP3 confers NLRP6-like ligand specificity .

• While NLRP3 responds to various cellular homeostasis disruptions, NLRP6 appears more specifically tuned to endolysosomal damage .

These differences highlight the importance of using specific antibodies when studying different inflammasome pathways, as the activation mechanisms and downstream effects may vary significantly.

What are the critical controls when using NLRP6 antibody in inflammasome research?

When conducting inflammasome research with NLRP6 antibody (HRP-conjugated), several critical controls should be implemented:

• Negative Controls: Include NLRP6-deficient samples (Nlrp6^-/- cells or tissues) to confirm antibody specificity.

• Positive Controls: Use samples with known NLRP6 expression (e.g., small intestine tissue) .

• Stimulus Controls: Compare results between wild-type Listeria monocytogenes and LLO-deficient (Δhly) strains, as the latter cannot activate NLRP6 .

• Cross-reactivity Controls: Test related NLR family proteins to ensure specificity.

• Isotype Controls: Include appropriate isotype-matched antibodies (Rabbit IgG for many NLRP6 antibodies) .

These controls help establish the specificity of observed signals and validate experimental findings, particularly important when investigating complex signaling cascades like inflammasome activation.

What tissue-specific optimizations are required for NLRP6 antibody in inflammation studies?

Tissue-specific optimizations for NLRP6 antibody detection vary based on the sample type:

For intestinal tissues (where NLRP6 is predominantly expressed):

• Standard fixation protocols are typically sufficient, though freshly prepared tissues yield better results.

• Antigen retrieval may be unnecessary or minimal due to higher expression levels.

For liver tissues (especially in disease models like schistosomiasis):

• More rigorous antigen retrieval is recommended with either:

  • TE buffer at pH 9.0 (preferred method)

  • Citrate buffer at pH 6.0 (alternative method)

• Extended primary antibody incubation (overnight at 4°C) may improve detection in fibrous liver tissues.

For cultured cells (like A549):

• Permeabilization optimization is crucial (0.1-0.3% Triton X-100 typically works well).

• Reduced antibody concentration may be necessary to minimize background.

These tissue-specific adjustments help maximize signal-to-noise ratio and ensure accurate detection of NLRP6 across different experimental systems.

How does NLRP6 contribute to hepatic immunopathology in disease models?

NLRP6 plays a significant role in hepatic immunopathology, particularly in the context of Schistosoma mansoni infection:

• NLRP6 is involved in IL-1β production and caspase-1 activation in response to soluble egg antigens (SEA) from S. mansoni.

• NLRP6 deficiency (Nlrp6^-/- mice) leads to:

  • Reduced periovular inflammation

  • Decreased collagen deposition in hepatic granulomas

  • Lower mRNA levels of α-SMA and IL-13

  • Altered chemokine and cytokine production

  • Modified macrophage and neutrophil recruitment into the liver

These findings reveal that NLRP6 is an essential component for schistosomiasis-associated pathology and fibrotic processes. The use of NLRP6 antibodies (including HRP-conjugated versions) in these disease models allows researchers to track expression levels and localization patterns during disease progression, providing insights into potential therapeutic interventions.

What is the role of NLRP6 in detecting endolysosomal damage and pathogen invasion?

Recent research has established that NLRP6 functions as a sensor for endolysosomal damage, whether caused by sterile triggers or bacterial pathogens:

• NLRP6 forms an inflammasome complex upon detecting endolysosomal disruption.

• This detection mechanism is particularly important for sensing bacterial pathogens like Listeria monocytogenes that enter the host cell cytosol.

• Unlike previously proposed ligands (LTA or poly(I:C)), endolysosomal damage appears to be the primary activator of NLRP6 .

• The NACHT domain of NLRP6 is the critical sensor region for this detection, while the LRR domain enhances but is not absolutely required for activity .

These findings have significant implications for understanding intestinal immunity and inflammatory disorders. HRP-conjugated NLRP6 antibodies are valuable tools for visualizing NLRP6 localization during endolysosomal damage and tracking its recruitment to sites of bacterial invasion.

How does NLRP6 expression vary across different cell types and tissues?

NLRP6 shows a distinctive expression pattern across tissues and cell types:

• High Expression: NLRP6 is predominantly expressed in primary intestinal epithelial cells (IECs).

• Low/No Expression: Mouse bone-marrow derived macrophages show little to no expression, even after priming with TLR ligands or interferon-γ (IFNγ) .

• Disease States: NLRP6 expression may be altered in various disease conditions, such as:

  • Increased expression in inflammatory bowel diseases

  • Modulated expression in fibrotic liver diseases

  • Co-regulation with NLRP3 in certain liver processes

• Species Differences: Expression patterns may vary between human, mouse, and rat tissues .

Understanding these expression patterns is critical when designing experiments with NLRP6 antibodies. Researchers should consider baseline expression levels in their target tissues and account for potential changes in disease models.

What are the optimal sample preparation protocols for NLRP6 detection in different applications?

Optimal sample preparation varies by application method:

For Western Blot:

• Extract proteins in RIPA buffer supplemented with protease inhibitors.

• Heat samples at 95°C for 5 minutes in reducing sample buffer.

• Load 20-50 μg of total protein per lane.

• Use PVDF membrane for better protein retention.

For Immunohistochemistry:

• Fix tissues in 10% neutral buffered formalin.

• Use TE buffer (pH 9.0) for antigen retrieval (preferred method).

• Alternative: use citrate buffer (pH 6.0) for antigen retrieval.

• Block with 5% normal serum from the same species as the secondary antibody .

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde for 15 minutes.

• Permeabilize with 0.1-0.3% Triton X-100 in PBS.

• Block with 1-5% BSA in PBS.

• Use appropriate fluorescent secondary antibodies for detection.

These protocols provide starting points but may require optimization for specific experimental conditions.

What strategies can address non-specific binding when using NLRP6 antibody in complex tissue samples?

Non-specific binding is a common challenge when using NLRP6 antibodies in complex tissues. Several strategies can minimize this issue:

• Extensive Blocking: Increase blocking time (1-2 hours) and use a combination of normal serum (5%) and BSA (1-3%).

• Antibody Titration: Carefully optimize antibody concentration, starting with higher dilutions (e.g., 1:500 for IHC) and adjusting as needed.

• Secondary Antibody Controls: Include controls omitting primary antibody to identify non-specific secondary antibody binding.

• Cross-Adsorption: Consider using cross-adsorbed secondary antibodies if working with multiple species.

• Endogenous Peroxidase Quenching: For HRP-conjugated antibodies, quench endogenous peroxidase activity by treating sections with 0.3% H₂O₂ in methanol for 30 minutes before blocking.

• Detergent Optimization: Adjust detergent concentration in wash buffers (0.05-0.1% Tween-20) to reduce background without compromising specific signals.

These approaches should be systematically tested to determine the optimal combination for each tissue type.

How can researchers optimize detection sensitivity for NLRP6 in low-expression scenarios?

When NLRP6 expression is low or difficult to detect, several optimization strategies can enhance sensitivity:

• Signal Amplification Systems:

  • For HRP-conjugated antibodies, employ tyramide signal amplification (TSA).

  • Use biotin-streptavidin amplification systems.

• Extended Incubation:

  • Increase primary antibody incubation time (overnight at 4°C).

  • Extend substrate development time for HRP detection.

• Sample Enrichment:

  • For cellular studies, consider using techniques to concentrate the protein of interest.

  • Immunoprecipitation followed by western blotting can enrich for NLRP6.

• Reduction of Background:

  • Use specialized blocking agents for problematic tissues.

  • Include protein A/G pre-adsorption steps to remove non-specific antibodies.

• Alternative Detection Systems:

  • Consider highly sensitive fluorescent detection methods for immunofluorescence.

  • Use chemiluminescent substrates with extended emission time for western blotting.

These approaches can significantly improve detection of low-abundance NLRP6, particularly important when studying cells or tissues where expression is naturally low or downregulated in disease states.

How can NLRP6 antibodies be used to study inflammasome assembly dynamics?

NLRP6 antibodies enable detailed investigation of inflammasome assembly dynamics through several advanced approaches:

• Co-immunoprecipitation Studies:

  • Use NLRP6 antibodies to pull down associated proteins like ASC and caspase-1.

  • Analyze the composition of NLRP6 inflammasome complexes under different stimulation conditions.

• Live Cell Imaging:

  • Combine antibody staining with fluorescently tagged ASC to track inflammasome speck formation.

  • Measure the kinetics of NLRP6 recruitment to ASC specks following stimulation.

• Proximity Ligation Assays:

  • Detect in situ interactions between NLRP6 and other inflammasome components.

  • Quantify interaction frequencies under different experimental conditions.

• Domain-specific Detection:

  • Use antibodies targeting different domains (PYD, NACHT, LRR) to study conformational changes during activation.

  • Track domain accessibility changes during the transition from inactive to active states.

These techniques can reveal the temporal and spatial dynamics of NLRP6 inflammasome assembly, particularly following endolysosomal damage or Listeria infection .

What are the best approaches for multiplexed detection of NLRP6 with other inflammasome components?

Multiplexed detection of NLRP6 with other inflammasome components requires careful planning:

These multiplexed approaches allow researchers to simultaneously visualize NLRP6 with other inflammasome components like ASC, caspase-1, and NLRP3, providing insights into their relative localization and potential interactions during inflammatory responses.

How can functional assays be combined with NLRP6 antibody detection to correlate expression with activity?

Correlating NLRP6 expression with its functional activity requires combining antibody detection with functional readouts:

• IL-1β/IL-18 Production:

  • Detect NLRP6 expression using antibodies while simultaneously measuring secreted IL-1β/IL-18 by ELISA.

  • Correlate expression levels with cytokine production in response to stimuli like bacterial infection.

• Caspase-1 Activity:

  • Use fluorescent caspase-1 substrates to measure activity while detecting NLRP6 localization.

  • FAM-FLICA (fluorochrome-labeled inhibitor of caspases) assays can be combined with immunofluorescence.

• Cell Death Assessments:

  • Combine NLRP6 immunostaining with cell death markers (Annexin V, propidium iodide).

  • Analyze how NLRP6 expression correlates with pyroptosis induction.

• Real-time Monitoring:

  • Use HRP-conjugated NLRP6 antibodies in live-cell compatible enzymatic assays.

  • Monitor changes in localization concurrent with functional readouts.

• Genetic Manipulation:

  • Compare wild-type and NLRP6-deficient cells/tissues using antibodies against downstream markers.

  • Rescue experiments with NLRP6 mutants can identify critical functional domains.

These integrated approaches establish direct links between NLRP6 expression patterns and functional outcomes, providing deeper mechanistic insights than either approach alone.