Sigirr Antibody

What is SIGIRR and what cellular functions does it perform?

SIGIRR (Single Ig and TIR domain containing) is a transmembrane protein also known as TIR8 or IL-1R8 that functions as a negative regulator of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways . The protein has a molecular mass of approximately 45.7 kilodaltons and contains a single extracellular immunoglobulin domain along with an intracellular Toll/IL-1R (TIR) domain . SIGIRR acts primarily as an inhibitory receptor that dampens inflammatory responses by interfering with the recruitment of adapter molecules to TLR and IL-1R complexes. This inhibitory function is crucial for maintaining immune homeostasis and preventing excessive inflammatory responses that could lead to tissue damage or inflammatory disorders.

SIGIRR is expressed in various tissues and cell types, with particularly significant expression in epithelial cells of the kidney, digestive tract, and respiratory system, as well as in certain immune cells including dendritic cells and B lymphocytes. Its expression patterns suggest tissue-specific regulatory roles in different physiological and pathological contexts.

What are the principal applications of SIGIRR antibodies in immunological research?

SIGIRR antibodies serve multiple crucial applications in immunological research, with Western blotting (WB) and immunohistochemistry (IHC) being the most commonly validated methods . In Western blot applications, SIGIRR antibodies typically require dilutions ranging from 1:500 to 1:1000 for optimal detection of the protein in tissue lysates such as mouse lung . For immunohistochemistry, dilutions of 1:50 to 1:500 are recommended, with successful detection demonstrated in human intrahepatic cholangiocarcinoma tissue and human ovary cancer tissue .

Additional validated applications include ELISA, immunocytochemistry (ICC), immunofluorescence (IF), and flow cytometry (FCM) . The choice of application depends on the specific research question, with WB being preferred for quantitative expression analysis, IHC for spatial localization in tissues, and flow cytometry for cell-specific expression patterns. Notably, some antibody clones have specific optimizations - for instance, certain monoclonal antibodies perform exceptionally well in flow cytometry applications for detecting surface SIGIRR expression.

How can researchers effectively validate SIGIRR antibody specificity?

Validating antibody specificity is crucial for ensuring reliable research results. For SIGIRR antibodies, a multi-step validation approach is recommended. Begin with positive and negative control samples - mouse lung tissue has been confirmed as a reliable positive control for Western blot applications . For negative controls, consider using tissues from SIGIRR knockout models or cell lines with SIGIRR knockdown via siRNA/shRNA.

Antigen competition assays provide another validation method, where pre-incubation of the antibody with purified SIGIRR protein or the immunizing peptide should abolish specific signals. Cross-reactivity testing across species is important, particularly as many SIGIRR antibodies show reactivity with human and mouse samples, while some also detect rat SIGIRR .

For definitive validation, consider using multiple antibodies targeting different epitopes of SIGIRR and compare the detection patterns. Consistent results across different antibodies strongly support specificity. Additionally, correlation of protein detection with mRNA expression data can provide further confidence in antibody specificity.

What are the optimal conditions for detecting SIGIRR in Western blot analyses?

The detection of SIGIRR in Western blot requires careful optimization of several parameters. Based on validated protocols, the following conditions yield optimal results:

For troubleshooting weak signals, extended primary antibody incubation (up to 48 hours at 4°C) can improve detection. Additionally, using freshly prepared samples is crucial, as SIGIRR protein stability can diminish with repeated freeze-thaw cycles. When analyzing specific tissues, mouse lung shows consistent SIGIRR expression and serves as an excellent positive control .

What methodological approaches optimize SIGIRR detection in immunohistochemistry?

Successful SIGIRR immunohistochemistry requires careful attention to several critical parameters:

Antigen retrieval is particularly important for SIGIRR detection, with TE buffer at pH 9.0 being the preferred method . Alternatively, citrate buffer at pH 6.0 can be used, though this may result in slightly reduced sensitivity. For formalin-fixed paraffin-embedded (FFPE) tissues, heat-induced epitope retrieval using a pressure cooker yields superior results compared to microwave methods.

The optimal antibody dilution range is 1:50 to 1:500, though this should be titrated for each specific tissue type and fixation method . Overnight incubation at 4°C generally produces more consistent staining compared to shorter incubations at room temperature. A polymer-based detection system typically provides better signal-to-noise ratio compared to avidin-biotin complex methods.

To minimize background staining, implement additional blocking steps including:

• 0.3% hydrogen peroxide in methanol for 10 minutes (to block endogenous peroxidase)

• Avidin/biotin blocking for 15 minutes each (if using biotin-based detection)

• 10% normal serum from the same species as the secondary antibody

Counterstaining with hematoxylin should be brief (1-2 minutes) to avoid masking weak SIGIRR signals. Always include serial sections stained with isotype control antibodies to confirm staining specificity.

How can researchers effectively troubleshoot non-specific binding with SIGIRR antibodies?

Non-specific binding is a common challenge when working with SIGIRR antibodies. A systematic troubleshooting approach includes:

For Western blot applications:

• Increase blocking stringency by extending blocking time to 2 hours or using a combination of 5% milk and 1% BSA

• Add 0.1-0.3% Tween-20 to antibody dilution buffers to reduce hydrophobic interactions

• Perform additional washing steps (5-6 washes of 10 minutes each) with 0.1% TBST

• Pre-adsorb the primary antibody with proteins from non-relevant species

• Titrate the antibody to find the highest dilution that still gives a specific signal

For immunohistochemistry applications:

• Use antigen retrieval optimization with a pH gradient test (pH 6.0, 8.0, and 9.0)

• Implement dual blocking with both protein blockers and commercial background reducing agents

• Consider using a mouse-on-mouse blocking kit when using mouse monoclonals on mouse tissue

• Decrease the concentration of the primary antibody while extending incubation time

• For tissues with high endogenous biotin, use a biotin blocking system or switch to a polymer-based detection method

In flow cytometry:

• Include dead cell discrimination dyes to eliminate non-specific binding to dead cells

• Use Fc receptor blocking reagents before antibody staining

• Perform absorption controls by pre-incubating the antibody with recombinant SIGIRR protein

How should researchers design experiments to investigate SIGIRR expression across different cell types?

Designing robust experiments to compare SIGIRR expression across cell types requires a multi-technique approach:

First, establish baseline expression using quantitative Western blot analysis with strictly controlled protein loading (verified by housekeeping proteins like β-actin or GAPDH) . This provides quantifiable data on relative expression levels. Follow this with flow cytometry to assess cell-surface versus intracellular SIGIRR distribution, which can vary significantly between cell types. Use fluorochrome-conjugated antibodies with appropriate isotype controls and implement compensation when multiplexing.

For spatial distribution analysis, perform immunofluorescence microscopy with co-staining for cell-type specific markers. This approach reveals both the cellular and subcellular localization patterns. To complement protein detection, incorporate RT-qPCR analysis of SIGIRR mRNA to correlate transcript levels with protein expression.

For comprehensive analysis, examine SIGIRR expression under both basal conditions and following relevant stimuli (e.g., TLR ligands, IL-1β) as SIGIRR expression can be dynamically regulated in response to inflammatory signals.

What are the recommended protocols for optimizing SIGIRR antibody concentration?

Optimizing SIGIRR antibody concentration requires a systematic titration approach tailored to each application:

For Western blot optimization:

• Prepare a serial dilution series of the antibody (e.g., 1:250, 1:500, 1:1000, 1:2000, 1:5000)

• Apply each dilution to identical blots containing both positive control samples (e.g., mouse lung tissue) and samples of interest

• Maintain all other conditions constant (blocking, incubation time, detection method)

• Evaluate signal-to-noise ratio at each concentration

• Select the highest dilution that produces a clear specific band with minimal background

For immunohistochemistry titration:

• Prepare a dilution series (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000)

• Apply to serial sections of positive control tissues (e.g., human intrahepatic cholangiocarcinoma or ovary cancer tissue)

• Maintain consistent antigen retrieval conditions (preferably TE buffer pH 9.0)

• Evaluate staining intensity, specificity, and background at each dilution

• Select the optimal dilution that maximizes specific staining while minimizing non-specific background

For flow cytometry optimization:

• Start with manufacturer recommendations for fluorochrome-conjugated antibodies

• Perform a broad range titration (e.g., 5μl, 2.5μl, 1μl, 0.5μl per million cells)

• Calculate staining index (mean positive signal - mean negative signal) / (2 × standard deviation of negative signal)

• Select the concentration with the highest staining index value

Document all optimization conditions in laboratory records to ensure reproducibility across experiments and between researchers.

What experimental controls are essential when using SIGIRR antibodies?

Implementing rigorous controls is critical for ensuring reliable interpretation of SIGIRR antibody-based experiments:

Essential negative controls:

• Primary antibody omission - Verifies detection system specificity

• Isotype control antibody - Controls for non-specific binding of immunoglobulin

• SIGIRR-knockout tissues/cells (when available) - Confirms absolute specificity

• SIGIRR-knockdown samples (siRNA/shRNA treated) - Alternative when knockout samples unavailable

• Pre-absorption with immunizing peptide - Verifies epitope-specific binding

Essential positive controls:

• Validated SIGIRR-expressing tissues (e.g., mouse lung for Western blot)

• Recombinant SIGIRR protein - Serves as reference standard

• Cell lines with documented SIGIRR expression - Provides consistent control samples

• SIGIRR-overexpressing transfected cells - Useful for determining antibody sensitivity

Application-specific controls:

• For Western blot: Molecular weight markers and loading controls (β-actin, GAPDH)

• For IHC/IF: Serial sections with secondary antibody only

• For flow cytometry: Fluorescence-minus-one (FMO) controls

• For multiplex assays: Single-stain controls for spectral compensation

Implementing both biological replicates (different samples) and technical replicates (same sample, repeated measures) strengthens the validity of experimental findings. When possible, validate key findings using multiple antibody clones targeting different SIGIRR epitopes.

How can researchers effectively use SIGIRR antibodies to investigate protein-protein interactions?

SIGIRR (IL-1R8/TIR8) participates in complex protein-protein interactions within immune signaling pathways. To investigate these interactions using SIGIRR antibodies, researchers should employ the following methodological approaches:

Co-immunoprecipitation (Co-IP) represents the gold standard for investigating SIGIRR interactions. Use a lysis buffer containing 1% NP-40 or 0.5% Triton X-100 with protease inhibitors to preserve protein complexes. Pre-clear lysates with protein A/G beads before immunoprecipitation to reduce non-specific binding. For Co-IP, antibodies should be carefully selected - use those validated for immunoprecipitation applications, typically those recognizing native epitopes rather than denatured ones.

Proximity ligation assay (PLA) offers an alternative for detecting protein interactions in situ. This technique generates fluorescent signals only when two proteins are within 40nm of each other, allowing visualization of SIGIRR interactions within cellular contexts. When implementing PLA, include appropriate controls including single primary antibody controls and non-interacting protein pair controls.

For investigating dynamic interactions, consider combining SIGIRR antibody approaches with techniques like FRET (Fluorescence Resonance Energy Transfer) or BiFC (Bimolecular Fluorescence Complementation). These methods require fluorescently tagged proteins but provide valuable insights into real-time interaction dynamics.

When analyzing interaction data, quantify co-precipitation results by normalizing the amount of co-precipitated protein to the amount of immunoprecipitated SIGIRR. For PLA, quantify the number of interaction spots per cell across multiple cells and fields.

What are the methodological approaches for quantifying SIGIRR expression in tissue samples?

Quantifying SIGIRR expression in tissue samples requires a multi-method approach for comprehensive and reliable data:

For Western blot quantification:

• Use gradient gels (4-15%) to ensure optimal resolution of the 45.7 kDa SIGIRR protein

• Include a standard curve of recombinant SIGIRR protein for absolute quantification

• Normalize SIGIRR band intensity to loading controls (β-actin or GAPDH)

• Use digital imaging and analysis software (ImageJ, Image Lab) for densitometry

• Present data as relative expression (fold change) compared to control samples or absolute quantities

For immunohistochemical quantification:

• Use automated staining platforms when possible to ensure consistency

• Standardize image acquisition parameters (exposure, gain, offset)

• Implement digital pathology approaches using software like QuPath or HALO

• Quantify both staining intensity (0-3+ scale) and percentage of positive cells

• Calculate H-scores (0-300) using the formula: Σ(intensity × percentage) for statistical comparisons

For the most robust analysis, compare SIGIRR protein levels with mRNA expression using parallel samples. This approach helps identify post-transcriptional regulatory mechanisms that may affect SIGIRR expression in different pathological contexts.

How can researchers address conflicting data regarding SIGIRR expression and function?

Conflicting data regarding SIGIRR expression and function is not uncommon in the literature. Addressing these discrepancies requires systematic analytical approaches:

First, conduct a comprehensive literature review to categorize contradictory findings and identify potential sources of variability. Common factors contributing to discrepancies include:

• Antibody clone differences - Different epitope recognition can yield varying results

• Species variations - Human, mouse, and rat SIGIRR may have distinct expression patterns

• Methodology differences - Western blot versus IHC versus flow cytometry

• Sample preparation variations - Fixation methods, buffer compositions

• Cell activation states - Baseline versus stimulated conditions

To directly address discrepancies:

• Reproduce contradictory experiments using identical methodology while carefully controlling variables. Document all experimental conditions meticulously.

• Implement orthogonal approaches - If Western blot and IHC results conflict, add flow cytometry and RT-qPCR to triangulate actual expression patterns.

• Consider post-translational modifications - SIGIRR undergoes glycosylation that affects antibody recognition; use deglycosylation experiments to standardize detection.

• Examine splice variants - Some antibodies may detect specific SIGIRR isoforms; use primers/antibodies targeting different domains to identify variant-specific expression.

• Validate with genetic approaches - Use CRISPR-Cas9 to generate SIGIRR knockout controls, or implement siRNA knockdown to confirm antibody specificity.

When publishing, transparently report all methodological details that could affect outcomes, and explicitly acknowledge limitations. Consider directly addressing known contradictions in the literature and providing reasoned explanations for discrepancies based on your methodological findings.

How can SIGIRR antibodies be utilized in multiplexed immunoassays?

Multiplexed immunoassays allow simultaneous detection of SIGIRR alongside other proteins, providing comprehensive insights into signaling pathways. For developing multiplexed assays involving SIGIRR antibodies, consider the following methodological approaches:

For multiplex immunofluorescence microscopy:

• Select SIGIRR antibodies from different host species than other target antibodies

• If using same-species antibodies, implement sequential staining with thorough blocking between rounds

• Use directly conjugated antibodies with spectrally distinct fluorophores

• Include controls for fluorophore bleed-through and cross-reactivity

• Employ multispectral imaging and unmixing algorithms for optimal signal separation

For multiplex flow cytometry:

• Carefully titrate each antibody in the panel individually before combining

• Use fluorophores with minimal spectral overlap for SIGIRR and co-markers

• Include fluorescence-minus-one (FMO) controls for each marker

• Implement compensation using single-stained controls

• Consider brightness of fluorophores relative to expected expression levels

For bead-based multiplex assays (e.g., Luminex):

• Validate SIGIRR antibody pairs (capture and detection) for specificity

• Test for cross-reactivity with other antibodies in the multiplex panel

• Establish standard curves using recombinant SIGIRR alongside other targets

• Optimize bead concentrations and sample dilutions to ensure linearity

• Implement spiking experiments to confirm accuracy in complex matrices

When analyzing multiplexed data, use dimensionality reduction techniques (tSNE, UMAP) for visualization and consider computational approaches like CITRUS (Cluster Identification, Characterization, and Regression) to identify biologically meaningful populations based on multiple parameters including SIGIRR expression.

What methods are available for studying SIGIRR phosphorylation and post-translational modifications?

Studying SIGIRR post-translational modifications (PTMs) requires specialized approaches beyond standard antibody applications:

For phosphorylation analysis:

• Use phospho-specific antibodies when available, typically requiring 1:250-1:500 dilutions

• Implement phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) during sample preparation

• Confirm specificity using lambda phosphatase treatment as a negative control

• Consider Phos-tag™ gel electrophoresis to separate phosphorylated from non-phosphorylated SIGIRR

• For sites without available antibodies, use immunoprecipitation followed by mass spectrometry

For glycosylation studies:

• Use enzymatic deglycosylation (PNGase F, Endo H) to confirm N-linked glycosylation

• Implement lectin blotting in parallel with SIGIRR antibody detection

• Compare molecular weight shifts before and after deglycosylation

• For comprehensive glycan analysis, combine immunoprecipitation with glycomics approaches

For ubiquitination analysis:

• Co-immunoprecipitate SIGIRR under denaturing conditions

• Probe with anti-ubiquitin antibodies

• Use proteasome inhibitors (MG132) during cell treatment to accumulate ubiquitinated species

• Consider tandem ubiquitin binding entity (TUBE) pulldowns followed by SIGIRR detection

When analyzing PTM data, always compare modified SIGIRR levels to total SIGIRR levels for accurate quantification of modification stoichiometry. Use appropriate positive controls for each modification type, such as EGF-stimulated cells for phosphorylation studies.

How can researchers leverage SIGIRR antibodies in disease model investigations?

SIGIRR dysregulation has been implicated in various pathological conditions, including inflammatory disorders, cancer, and infectious diseases. Leveraging SIGIRR antibodies in disease models requires careful experimental design:

For inflammatory disease models:

• Implement time-course analyses to track SIGIRR expression changes during disease progression

• Combine IHC spatial mapping with quantitative Western blot analysis

• Correlate SIGIRR levels with inflammatory markers (cytokines, chemokines)

• Use flow cytometry to identify cell populations with altered SIGIRR expression

• Consider single-cell approaches to detect heterogeneity in SIGIRR regulation

For cancer research applications:

• Compare SIGIRR expression between tumor and adjacent normal tissues

• Correlate expression with clinical parameters and survival outcomes

• Investigate SIGIRR in tumor microenvironment components (immune infiltrates)

• Examine relationship between SIGIRR and established oncogenic pathways

• Use patient-derived xenografts to maintain tumor heterogeneity

For infectious disease models:

• Analyze SIGIRR expression changes during pathogen infection cycles

• Investigate pathogen-mediated SIGIRR regulation mechanisms

• Correlate SIGIRR levels with pathogen burden and inflammatory responses

• Compare effects in wild-type versus SIGIRR-deficient models

• Consider therapeutic targeting approaches based on SIGIRR pathway manipulation

When designing translational studies, implement tissue microarrays for high-throughput screening across multiple patient samples. Combine with multiplex immunofluorescence to simultaneously detect SIGIRR alongside disease-specific markers. For mechanistic insights, complement antibody-based detection with functional assays measuring downstream signaling events (NF-κB activation, cytokine production) in the presence of SIGIRR-modulating agents.