IL1RAPL1 Antibody, FITC conjugated

What is IL1RAPL1 and why is it important in neuroscience research?

IL1RAPL1 (Interleukin-1 receptor accessory protein-like 1) is a member of the interleukin 1 receptor family that shares approximately 52% homology with IL-1 receptor accessory protein (IL1RAcP). The protein contains three extracellular immunoglobulin-like domains, a transmembrane domain, an intracellular Toll/IL-1R (TIR) domain, and a unique C-terminal tail of 150 amino acids. IL1RAPL1 is predominantly expressed in the brain and is enriched at excitatory synapses, particularly in the postsynaptic compartment . Its importance stems from its involvement in synapse formation and function, with mutations in the IL1RAPL1 gene associated with cognitive impairments ranging from non-syndromic X-linked intellectual disability to autism . Studies using Il1rapl1 knockout mice have demonstrated impaired associative learning and synaptic defects, including decreased dendritic spine density and altered synaptic plasticity across multiple brain regions .

How does the FITC conjugation affect the application range of IL1RAPL1 antibody?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent labeling of the IL1RAPL1 antibody, eliminating the need for secondary antibody incubation in immunofluorescence applications. This conjugation enables direct visualization of IL1RAPL1 in fluorescence microscopy, flow cytometry, and other fluorescence-based techniques. The FITC conjugate emits green fluorescence (peak emission ~520 nm) when excited with blue light (peak excitation ~495 nm), making it compatible with standard FITC filter sets . While the conjugation expands the application range to include direct detection methods, it's important to note that the bulky FITC molecule could potentially affect antibody binding in some contexts, particularly in densely packed cellular structures or when epitope accessibility is limited.

What are the target epitopes of commercially available IL1RAPL1 antibodies?

The commercially available IL1RAPL1 antibodies target different epitopes of the protein. For example, the FITC-conjugated polyclonal antibody from Qtonics (QA26211) targets the recombinant Human Interleukin-1 receptor accessory protein-like 1 protein fragment spanning amino acids 564-679 . Similarly, the antibody available from antibodies-online (ABIN7156735) also targets the amino acid region 564-679 . Other commercially available antibodies may target different regions, such as the extracellular domains, the TIR domain, or the unique C-terminal region. The epitope location is crucial for experimental design, as antibodies targeting different domains may yield different results depending on protein conformation, post-translational modifications, or interactions with binding partners.

What are the optimal protocols for using IL1RAPL1 Antibody, FITC conjugated in neuronal culture immunofluorescence studies?

For optimal immunofluorescence staining of cultured neurons using IL1RAPL1 Antibody, FITC conjugated:

• Fixation: Fix neurons (typically DIV14-21) with 4% paraformaldehyde in PBS for 15 minutes at room temperature.

• Permeabilization: Permeabilize with 0.1% Triton X-100 in PBS for 5-10 minutes.

• Blocking: Block with 5% normal goat serum (NGS) in PBS for 1 hour at room temperature.

• Primary antibody: Dilute FITC-conjugated IL1RAPL1 antibody to 1:100-1:500 in blocking solution and incubate overnight at 4°C in a humid chamber protected from light.

• Washing: Wash 3x with PBS for 5 minutes each.

• Counterstaining: Optional counterstaining with DAPI (1:5000) for nuclei and phalloidin (1:1000) for F-actin/dendritic spines.

• Mounting: Mount with anti-fade mounting medium.

When studying synaptic localization, co-staining with synaptic markers such as PSD-95 (post-synaptic) or synaptophysin/VGLUT1 (pre-synaptic) can provide valuable context . For optimal visualization, use confocal microscopy with appropriate filter settings for FITC (excitation ~495 nm, emission ~520 nm). To assess specificity, include controls using IL1RAPL1 knockout neurons or IL1RAPL1-depleted neurons via shRNA.

How can I optimize Western blot protocols for detecting IL1RAPL1 using specific antibodies?

For optimal Western blot detection of IL1RAPL1:

• Sample preparation:

  • Extract proteins from neuronal cultures or brain tissue using RIPA buffer supplemented with protease inhibitors

  • For membrane proteins like IL1RAPL1, add 0.5% sodium deoxycholate to improve solubilization

• Protein separation:

  • Use 8-10% SDS-PAGE gels as IL1RAPL1 is approximately 115 kDa

  • Load 20-50 μg of total protein per lane

• Transfer conditions:

  • Transfer to PVDF membrane (preferred over nitrocellulose for this protein)

  • Use wet transfer at 30V overnight at 4°C for optimal transfer of large proteins

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • For unconjugated IL1RAPL1 antibodies: dilute 1:500-1:1000 in blocking buffer, incubate overnight at 4°C

  • For HRP-conjugated secondary antibody: dilute 1:5000-1:10000, incubate for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Expected band size: ~115 kDa for wild-type IL1RAPL1, ~108 kDa for Δex6 mutant

• Controls:

  • Positive control: overexpressed IL1RAPL1 in HEK293 cells

  • Negative control: lysate from IL1RAPL1 knockout tissue or cells transfected with empty vector

  • Loading control: β-actin or GAPDH

When analyzing mutant forms of IL1RAPL1, be aware that mutations may affect protein stability and expression levels. For instance, the Δex6 and C31R mutations lead to decreased protein stability (~75% and ~60% reduction respectively compared to wild-type) .

What controls should be included when using IL1RAPL1 antibodies for immunofluorescence experiments?

When conducting immunofluorescence experiments with IL1RAPL1 antibodies, include the following controls:

• Negative Controls:

  • Primary antibody omission: Incubate samples with secondary antibody only (for unconjugated primary antibodies) or buffer only (for conjugated antibodies) to assess non-specific binding

  • Tissue/cells lacking IL1RAPL1 expression: IL1RAPL1 knockout neurons or shRNA-mediated knockdown cells

  • Isotype control: Use an irrelevant antibody of the same isotype (IgG) and host species (rabbit) at the same concentration

• Specificity Controls:

  • Peptide competition: Pre-incubate the antibody with excess immunogenic peptide (amino acids 564-679 for the FITC-conjugated antibody ) before staining

  • Correlation with other IL1RAPL1 antibodies: Compare staining pattern with antibodies targeting different IL1RAPL1 epitopes

• Technical Controls:

  • Positive control: Cells overexpressing IL1RAPL1 (e.g., transfected neurons or HEK293 cells)

  • Dual-labeling with established synaptic markers: Co-stain with pre-synaptic (synaptophysin, VGLUT1) and post-synaptic (PSD-95) markers to confirm synaptic localization

  • Counterstaining with nuclear and cytoskeletal markers: DAPI for nuclei and phalloidin for F-actin to provide structural context

• Validation Controls:

  • Functional correlation: Compare antibody detection with functional assays such as electrophysiology recordings to correlate protein detection with functional states

  • Alternative detection methods: Validate findings with in situ hybridization for IL1RAPL1 mRNA or with tagged recombinant IL1RAPL1 expression

These controls ensure reliable and interpretable results when investigating IL1RAPL1 localization and expression in neuronal systems.

How can IL1RAPL1 Antibody, FITC conjugated be used to investigate synaptic defects in intellectual disability models?

The FITC-conjugated IL1RAPL1 antibody serves as a powerful tool for investigating synaptic defects in intellectual disability models through several methodologies:

• Quantitative Synapse Analysis:

  • Perform high-resolution confocal imaging of neurons stained with FITC-IL1RAPL1 antibody and co-stained with pre-synaptic (VGLUT1) and post-synaptic (PSD-95) markers

  • Quantify synaptic density, size, and colocalization indices in control versus intellectual disability models

  • Compare wild-type IL1RAPL1 distribution with mutant forms (e.g., Δex6, C31R) known to cause intellectual disability

• Live Imaging Applications:

  • Use the antibody to label surface IL1RAPL1 in live neurons when studying receptor trafficking and dynamics

  • Combine with FM4-64 dye labeling to assess synaptic vesicle recycling and correlate with IL1RAPL1 localization

• Molecular Interaction Studies:

  • Investigate colocalization between IL1RAPL1 and its binding partners such as PTPδ (pre-synaptic) and RhoGAP2 (post-synaptic)

  • Implement Proximity Ligation Assay (PLA) with the FITC-conjugated antibody and antibodies against interaction partners to visualize protein-protein interactions in situ

• Comparative Analysis Across Models:

  • Apply standardized staining protocols across different intellectual disability models (genetic mutations, environmental factors)

  • Create quantitative profiles of synaptic alterations specific to IL1RAPL1-associated intellectual disability

  • Develop a comparative matrix of synaptic phenotypes across different forms of intellectual disability

Research has shown that IL1RAPL1 regulates excitatory synapse formation through its interaction with pre-synaptic PTPδ and post-synaptic RhoGAP2 . In intellectual disability models, mutations in IL1RAPL1 (such as Δex6 and C31R) fail to induce pre- and post-synaptic differentiation, unlike wild-type IL1RAPL1 . The FITC-conjugated antibody allows direct visualization of these defects, enabling quantitative assessment of synaptic alterations in various intellectual disability models.

What are the methodological approaches for investigating IL1RAPL1 interaction with PTPδ and RhoGAP2 using antibody-based techniques?

Several antibody-based methodological approaches can be employed to investigate IL1RAPL1 interactions with PTPδ and RhoGAP2:

• Co-immunoprecipitation (Co-IP):

  • Use anti-IL1RAPL1 antibody for immunoprecipitation from neuronal lysates

  • Detect PTPδ or RhoGAP2 in the precipitated complex by Western blotting

  • For reverse Co-IP, immunoprecipitate with anti-PTPδ or anti-RhoGAP2 and probe for IL1RAPL1

  • Research shows IL1RAPL1 TIR domains interact with RhoGAP2, which is localized at the excitatory post-synaptic density

• Proximity Ligation Assay (PLA):

  • Incubate fixed neurons with IL1RAPL1 antibody and antibodies against PTPδ or RhoGAP2

  • Apply species-specific PLA probes, ligase, and polymerase

  • Visualize interaction signals as fluorescent dots only where proteins are in close proximity (<40 nm)

  • Quantify interaction sites per cell or per dendritic segment

• FRET/FLIM Analysis:

  • Use FITC-conjugated IL1RAPL1 antibody as donor and a red-fluorescent labeled antibody against PTPδ or RhoGAP2 as acceptor

  • Measure FRET efficiency to determine molecular proximity

  • Calculate distance between proteins based on FRET parameters

• Bimolecular Fluorescence Complementation (BiFC):

  • Express IL1RAPL1 fused to one half of a fluorescent protein and PTPδ or RhoGAP2 fused to the complementary half

  • Reconstituted fluorescence occurs only upon protein interaction

  • Use antibodies to enhance detection or verify expression

• Domain-Specific Interaction Mapping:

  • Generate constructs expressing specific domains of IL1RAPL1 (e.g., TIR domain, amino acids 403-562; C-terminal fragments, amino acids 560-696)

  • Perform pull-down assays with these constructs and detect interaction partners

  • Research has shown that the TIR domain and the C-terminal fragments (amino acids 560-696 and 608-684) of IL1RAPL1 can precipitate RhoGAP2

• Functional Validation:

  • Conduct electrophysiological recordings following antibody-mediated disruption of IL1RAPL1 interactions

  • Correlate interaction disruption with synaptic function alterations

These methodological approaches have revealed that IL1RAPL1 forms a trans-synaptic interaction with PTPδ through its extracellular domain, while its intracellular TIR domain interacts with RhoGAP2 . These interactions are critical for proper excitatory synapse formation and function.

How does IL1RAPL1 antibody staining pattern differ between wild-type and intellectual disability-associated mutations?

The staining pattern of IL1RAPL1 antibodies reveals significant differences between wild-type and intellectual disability-associated mutant forms:

• Subcellular Localization Differences:

  • Wild-type IL1RAPL1: Localizes primarily to dendritic spines and excitatory synapses, with enrichment at the postsynaptic density. Shows punctate pattern along dendrites colocalizing with PSD-95 .

  • Δex6 Mutant: Shows diffuse distribution throughout dendrites rather than synaptic enrichment, indicating mislocalization. Many Δex6 mutant proteins fail to reach the cell surface and accumulate in the endoplasmic reticulum .

  • C31R Mutant: Retains some ability to localize to synapses but shows reduced clustering at synaptic sites compared to wild-type protein .

• Expression Level Variations:

  • Wild-type IL1RAPL1: Shows stable expression with clear punctate pattern.

  • Δex6 Mutant: Exhibits approximately 75% reduction in protein expression compared to wild-type, leading to weaker immunofluorescence signal .

  • C31R Mutant: Shows approximately 60% reduction in protein expression, resulting in diminished antibody staining intensity .

• Colocalization with Synaptic Markers:

  • Wild-type IL1RAPL1: Strong colocalization with excitatory synapse markers (VGLUT1, PSD-95) but not with inhibitory synapse markers (VGAT) .

  • Mutant Forms: Significantly reduced colocalization with excitatory synapse markers compared to wild-type, indicating failure to induce or maintain proper synaptic structures .

• Partner Protein Recruitment:

  • Wild-type IL1RAPL1: Antibody staining shows colocalization with RhoGAP2 in dendritic spines .

  • Mutant Forms: Reduced colocalization with RhoGAP2, consistent with the mutants' impaired ability to recruit RhoGAP2 to synapses .

• Pattern Changes in Activity-Dependent Experiments:

  • Wild-type IL1RAPL1: In neurons stimulated with elevated K+ (90 mM KCl), antibody staining shows increased clustering at active synapses, correlating with FM4-64 labeling of active presynaptic terminals .

  • Mutant Forms: Fail to show activity-dependent redistribution, reflecting their inability to participate in synapse maturation and plasticity .

These differential staining patterns provide valuable insight into the mechanisms by which IL1RAPL1 mutations lead to intellectual disability, highlighting defects in protein stability, subcellular trafficking, and synaptic recruitment of interaction partners.

What statistical approaches are recommended for analyzing IL1RAPL1 immunofluorescence data in comparative studies?

When analyzing IL1RAPL1 immunofluorescence data in comparative studies, the following statistical approaches are recommended:

• Quantification Parameters:

  • Synaptic density: Count IL1RAPL1-positive puncta per unit length of dendrite (typically per 10 μm)

  • Colocalization metrics: Calculate Manders' overlap coefficient or Pearson's correlation coefficient between IL1RAPL1 and synaptic markers

  • Fluorescence intensity: Measure integrated density or mean gray value of IL1RAPL1 signal at synapses

  • Morphological parameters: Analyze size, shape, and distribution of IL1RAPL1-positive structures

• Statistical Tests for Group Comparisons:

  • For normally distributed data: Student's t-test (two groups) or one-way ANOVA with post-hoc tests (multiple groups)

  • For non-normally distributed data: Mann-Whitney U test (two groups) or Kruskal-Wallis with post-hoc tests (multiple groups)

  • For paired comparisons (e.g., treated vs. untreated neurons from same culture): Paired t-test or Wilcoxon signed-rank test

• Advanced Statistical Methods:

  • Multi-factor ANOVA: For experiments with multiple independent variables (e.g., genotype, treatment, time point)

  • Mixed-effects models: For nested data structures (e.g., multiple neurons per animal, multiple dendrites per neuron)

  • ANCOVA: To account for covariates like neuron size or developmental stage

• Sample Size and Power Considerations:

  • Minimum sample sizes: Analyze at least 15-20 neurons per condition from at least 3 independent cultures

  • Power analysis: Use preliminary data to determine sample size needed for 80% power at α=0.05

  • Biological replicates: Ensure findings are reproducible across multiple independent experiments

• Controls for Quantitative Analysis:

  • Background subtraction: Subtract signal from regions without specific staining

  • Normalization strategies: Normalize IL1RAPL1 signal to total dendritic area or to housekeeping protein levels

  • Internal controls: Include wild-type and IL1RAPL1-deficient samples in each experiment as reference points

• Visualization of Results:

  • Representative images: Present typical examples accompanied by quantitative data

  • Combine techniques: Show colocalization as scatterplots plus overlaid images

  • Scale bars: Include appropriate scale bars (typically 5-10 μm for dendrite segments)

For example, when comparing wild-type IL1RAPL1 to mutant forms (Δex6 or C31R), quantify the colocalization with pre-synaptic markers like VGLUT1. Research has shown that while wild-type IL1RAPL1 significantly increases excitatory pre-synaptic marker density, the Δex6 and C31R mutants fail to induce this effect . Statistical analysis of such data should account for cell-to-cell variability and include appropriate controls.

How can I address potential false positives and negatives when using IL1RAPL1 antibodies in brain tissue from intellectual disability models?

Addressing false positives and negatives when using IL1RAPL1 antibodies in intellectual disability models requires a systematic approach:

• Antibody Validation Strategies:

  • Epitope mapping: Confirm antibody specificity for the intended epitope (amino acids 564-679 for FITC-conjugated antibody)

  • Cross-reactivity testing: Test antibody against brain tissue from IL1RAPL1 knockout animals

  • Signal verification: Validate signals using multiple antibodies targeting different IL1RAPL1 epitopes

  • Peptide competition: Pre-absorb antibody with immunizing peptide to block specific binding

• Technical Controls to Minimize False Positives:

  • Autofluorescence control: Include unstained sections to identify natural tissue fluorescence

  • Secondary-only control: Omit primary antibody to detect non-specific secondary antibody binding

  • Isotype control: Use irrelevant antibodies of same isotype and host species at equivalent concentration

  • Absorption controls: Pre-absorb antibody with the antigen prior to staining

• Strategies to Minimize False Negatives:

  • Antigen retrieval optimization: Test multiple antigen retrieval methods (heat-induced, enzymatic)

  • Fixation testing: Compare different fixation protocols (PFA concentrations, duration)

  • Signal amplification: Use tyramide signal amplification or other enhancement methods for low-abundance targets

  • Positive control tissues: Include samples known to express high levels of IL1RAPL1

• Analytical Approaches:

  • Thresholding techniques: Use objective methods like Otsu's method rather than subjective manual thresholding

  • Signal-to-noise ratio calculation: Quantify signal relative to background for accurate comparison

  • Z-stack imaging: Collect multiple optical sections to ensure complete sampling of the tissue

  • Blind analysis: Have images analyzed by researchers blinded to experimental conditions

• Confirmation with Orthogonal Methods:

  • mRNA validation: Correlate protein detection with mRNA expression using in situ hybridization

  • Functional correlation: Relate antibody staining patterns to electrophysiological recordings

  • Alternative detection methods: Confirm findings using tagged recombinant IL1RAPL1 in rescue experiments

• Advanced Quality Control:

  • Lot-to-lot validation: Test each new antibody lot against previous lots

  • Standardized positive controls: Include identical positive control samples across all experiments

  • Quantitative sensitivity testing: Determine detection limits using dilution series of recombinant protein

In intellectual disability research, it's particularly important to validate findings across multiple models. For example, when studying the C31R mutation, which reduces protein stability by approximately 60% , researchers should calibrate detection sensitivity to ensure that reduced expression is not misinterpreted as complete absence of the protein.

What are the key considerations when comparing IL1RAPL1 localization data between different experimental models of intellectual disability?

When comparing IL1RAPL1 localization data between different experimental models of intellectual disability, several key considerations should be addressed:

• Model System Characteristics:

  • Species differences: Consider evolutionary conservation of IL1RAPL1 when comparing across species (mouse vs. human neurons)

  • Cell type specificity: Account for differences between primary neurons, iPSC-derived neurons, and cell lines

  • Developmental stage: Normalize for neuronal maturity as IL1RAPL1 expression and localization change during development

  • Regional variations: Compare equivalent brain regions or neuronal subtypes across models

• Technical Standardization:

  • Fixation protocols: Use identical fixation methods across all samples (e.g., 4% PFA for 15 minutes)

  • Antibody conditions: Standardize antibody concentration, incubation time, and temperature

  • Imaging parameters: Maintain consistent microscope settings, exposure times, and resolution

  • Image processing: Apply identical processing algorithms to all datasets

• Controls and Calibration:

  • Internal standards: Include wild-type controls in each experiment

  • Cross-model calibration: Process representative samples from different models simultaneously

  • Reference markers: Co-stain with invariant synaptic markers for normalization

  • Biological replicates: Analyze multiple independent samples for each model

• Phenotypic Characterization Matrix:

  Parameter	Wild-type	Mutation 1 (e.g., Δex6)	Mutation 2 (e.g., C31R)	Other Models
  Protein expression level	Baseline	~25% of WT	~40% of WT	Model-specific
  Synaptic localization	Enriched	Diffuse	Partial	Model-specific
  VGLUT1 colocalization	High	Low	Low	Model-specific
  PSD-95 clustering	Enhanced	No effect	No effect	Model-specific
  Dendritic spine density	Increased	Unchanged	Unchanged	Model-specific

• Functional Correlations:

  • Electrophysiological parameters: Correlate IL1RAPL1 localization with sEPSC frequency and amplitude

  • Behavioral outcomes: Relate cellular phenotypes to behavioral deficits in animal models

  • Molecular interactions: Compare binding partner recruitment across models

• Data Integration Approaches:

  • Multiparametric analysis: Consider multiple aspects of IL1RAPL1 biology simultaneously

  • Machine learning classification: Use computational methods to identify patterns across models

  • Systems biology integration: Place IL1RAPL1 alterations in the context of broader synaptic networks

• Translational Relevance:

  • Human validation: Confirm findings in human patient-derived samples when possible

  • Phenotypic severity correlation: Relate molecular alterations to clinical severity

  • Intervention response: Compare how different models respond to therapeutic interventions

Research has shown that different IL1RAPL1 mutations affect protein function through distinct mechanisms. For example, while the Δex6 mutation leads to protein instability and mislocalization, the C31R mutation primarily affects interaction with PTPδ without dramatically altering localization . A comprehensive comparison framework helps distinguish mutation-specific effects from general consequences of IL1RAPL1 dysfunction across different intellectual disability models.

What are common pitfalls when using IL1RAPL1 Antibody, FITC conjugated, and how can they be addressed?

Common pitfalls when using IL1RAPL1 Antibody, FITC conjugated and their solutions include:

• High Background/Non-specific Staining:

  • Pitfall: Diffuse fluorescence throughout sample obscuring specific IL1RAPL1 signal

  • Solutions:

    • Optimize blocking (increase BSA/serum concentration to 5-10%)

    • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce hydrophobic interactions

    • Pre-absorb antibody with tissue lysate from IL1RAPL1 knockout samples

    • Reduce antibody concentration (try serial dilutions from 1:100 to 1:1000)

    • Include 0.1-0.3M NaCl in wash buffers to increase stringency

• Photobleaching of FITC Signal:

  • Pitfall: Rapid fading of fluorescence during imaging

  • Solutions:

    • Use anti-fade mounting media containing DABCO or PPD

    • Minimize exposure to excitation light during microscopy

    • Capture images quickly or use time-lapse protocols with minimal illumination

    • Consider switching to more photostable fluorophore-conjugated IL1RAPL1 antibodies (if available)

• Inadequate Tissue Penetration:

  • Pitfall: Signal limited to tissue surface in thicker sections

  • Solutions:

    • Optimize permeabilization (extend Triton X-100 treatment to 30-60 minutes)

    • Use thinner tissue sections (30 μm or less)

    • Implement antigen retrieval protocols (citrate buffer pH 6.0, 95°C for 10 minutes)

    • Extend antibody incubation time to 48-72 hours at 4°C for thick sections

• Signal Variability Across Experiments:

  • Pitfall: Inconsistent staining intensity between experimental batches

  • Solutions:

    • Aliquot antibody upon receipt to avoid freeze-thaw cycles

    • Include standard positive control in each experiment

    • Standardize all protocol parameters (fixation time, antibody concentration)

    • Process control and experimental samples simultaneously

• Autofluorescence Interference:

  • Pitfall: Tissue autofluorescence in the FITC channel confounding results

  • Solutions:

    • Perform autofluorescence quenching (0.1% Sudan Black B in 70% ethanol for 20 minutes)

    • Use spectral unmixing during confocal microscopy

    • Consider switching to far-red conjugated antibodies if available

    • Implement image processing to subtract autofluorescence

• Epitope Masking:

  • Pitfall: Inability to detect IL1RAPL1 due to protein interactions or conformational changes

  • Solutions:

    • Try multiple antigen retrieval methods (heat-induced, enzymatic, pH variations)

    • Test different fixation protocols (shorter fixation time, lower PFA concentration)

    • Use antibodies targeting different epitopes of IL1RAPL1

    • Consider native-protein-preserving fixation methods for conformationally sensitive epitopes

• Specificity Concerns:

  • Pitfall: Inability to distinguish between specific and non-specific signals

  • Solutions:

    • Validate with IL1RAPL1 knockout or knockdown controls

    • Perform peptide competition experiments

    • Compare staining pattern with multiple IL1RAPL1 antibodies

    • Correlate with IL1RAPL1 mRNA expression data

Implementing these solutions will help ensure reliable and reproducible results when using FITC-conjugated IL1RAPL1 antibodies for neurobiological research.

How can I validate the specificity of IL1RAPL1 antibody in studies of protein-protein interactions at synapses?

Validating the specificity of IL1RAPL1 antibody for protein-protein interaction studies at synapses requires a multi-layered approach:

• Genetic Validation:

  • Knockout Controls: Compare staining between wild-type and IL1RAPL1 knockout tissues

  • Knockdown Verification: Analyze tissues with shRNA-mediated IL1RAPL1 depletion

  • Rescue Experiments: Restore expression with tagged IL1RAPL1 constructs resistant to shRNA and verify antibody co-localization with the tag

• Biochemical Validation:

  • Western Blot Analysis: Confirm antibody detects a single band of appropriate molecular weight (~115 kDa)

  • Immunoprecipitation Specificity: Use antibody for IP followed by mass spectrometry to verify IL1RAPL1 enrichment

  • Peptide Competition: Pre-incubate antibody with immunogenic peptide (amino acids 564-679) before staining

• Cross-Antibody Validation:

  • Multiple Antibody Comparison: Compare staining patterns using antibodies targeting different IL1RAPL1 epitopes

  • Correlation Analysis: Quantify overlap between different antibody signals (Pearson's coefficient > 0.8 indicates high reliability)

  • Epitope Mapping: Use deletion constructs to confirm epitope specificity

• Interaction-Specific Validation:

  • Proximity Ligation Assays (PLA): Perform PLA between IL1RAPL1 and interaction partners (PTPδ, RhoGAP2)

  • FRET Analysis: Measure FRET between labeled IL1RAPL1 antibody and antibodies against interaction partners

  • Domain-Specific Controls: Compare interaction signals using IL1RAPL1 mutants lacking specific interaction domains:

    • IL1RAPL1▵N (lacking extracellular domain): Should eliminate PTPδ interaction

    • IL1RAPL1▵C (lacking C-terminus): Should eliminate RhoGAP2 interaction

• Subcellular Localization Verification:

  • Super-Resolution Microscopy: Use techniques like STORM or STED to precisely localize IL1RAPL1 relative to synaptic partners

  • Immuno-Electron Microscopy: Confirm ultrastructural localization at synapses

  • Subcellular Fractionation: Verify antibody detects IL1RAPL1 in synaptosomal but not non-synaptic fractions

• Functional Validation:

  • Antibody Perturbation: Apply antibody to live neurons to disrupt interactions and monitor functional effects

  • Correlation with Function: Compare antibody-detected interactions with electrophysiological parameters

  • Activity-Dependent Changes: Verify expected changes in interactions following neuronal stimulation

• Cross-Species Validation:

  • Conservation Analysis: Compare detection patterns across species with known IL1RAPL1 sequence conservation

  • Heterologous Systems: Test antibody in non-neuronal cells expressing recombinant IL1RAPL1 and partners

Research has shown that IL1RAPL1 interacts with PTPδ through its extracellular domain and with RhoGAP2 through its intracellular TIR domain . Proper validation ensures that antibody-detected interactions accurately reflect these molecular associations at synapses.

What emerging techniques might enhance the application of IL1RAPL1 antibodies in neurodevelopmental disorder research?

Several emerging techniques show promise for enhancing IL1RAPL1 antibody applications in neurodevelopmental disorder research:

• Advanced Imaging Technologies:

  • Expansion Microscopy: Physical expansion of samples to achieve super-resolution imaging of IL1RAPL1 at synapses using standard confocal microscopy

  • Light-Sheet Microscopy: Rapid volumetric imaging of IL1RAPL1 distribution across entire brain regions with minimal photobleaching

  • STORM/PALM Super-Resolution: Nanoscale localization of IL1RAPL1 relative to synaptic proteins, revealing precise spatial organization

  • Lattice Light-Sheet: Live imaging of antibody-labeled IL1RAPL1 dynamics in intact tissue with reduced phototoxicity

• Single-Cell Analysis Integration:

  • Spatial Transcriptomics + Immunofluorescence: Correlate IL1RAPL1 protein localization with transcriptome-wide expression patterns

  • Mass Cytometry (CyTOF): Multiplex IL1RAPL1 with dozens of other neuronal markers for comprehensive phenotyping

  • Single-Cell Western Blot: Analyze IL1RAPL1 expression variability across individual neurons within heterogeneous populations

• Functional Antibody Applications:

  • Optogenetic Antibodies: Light-activated antibodies that can temporally control IL1RAPL1 interactions

  • CRISPR-Based Tagging: Endogenous tagging of IL1RAPL1 for live visualization without antibody limitations

  • Intrabodies: Genetically encoded antibody fragments that can track IL1RAPL1 in living neurons

• High-Throughput Screening Platforms:

  • Microfluidic Neuron Culture Systems: Screen compounds that modify IL1RAPL1 localization or function

  • Automated High-Content Imaging: Quantify IL1RAPL1 phenotypes across thousands of neurons under various conditions

  • CRISPR Screens: Identify genetic modifiers of IL1RAPL1 dysfunction in intellectual disability models

• Human iPSC-Derived Models:

  • Brain Organoids: Study IL1RAPL1 in 3D human neural tissues with complex architecture

  • Patient-Derived Neurons: Compare IL1RAPL1 localization and function between neurons derived from patients and healthy controls

  • Isogenic iPSC Lines: Examine effects of specific IL1RAPL1 mutations in identical genetic backgrounds

• In Vivo Applications:

  • Viral Delivery of Tagged Antibody Fragments: Monitor IL1RAPL1 in intact circuits in vivo

  • Cranial Window Imaging: Longitudinal studies of IL1RAPL1 dynamics during development and in disease models

  • Nanobody-Based Probes: Smaller antibody-like probes with improved tissue penetration and reduced immunogenicity

• Translational Applications:

  • PET-Tracers Based on IL1RAPL1 Antibodies: Non-invasive imaging of IL1RAPL1 expression in human brain

  • Antibody-Drug Conjugates: Targeted delivery of therapeutic compounds to IL1RAPL1-expressing neurons

  • IL1RAPL1-Targeted Gene Therapy: Antibody-guided delivery of genetic interventions to affected neuronal populations

These emerging techniques promise to provide unprecedented insights into how IL1RAPL1 dysfunction contributes to intellectual disability and autism spectrum disorders, potentially opening new avenues for therapeutic intervention targeting this critical synaptic protein.

How might IL1RAPL1 antibodies contribute to the development of targeted therapeutics for intellectual disabilities?

IL1RAPL1 antibodies could significantly contribute to the development of targeted therapeutics for intellectual disabilities through several innovative approaches:

• Diagnostic and Patient Stratification Applications:

  • Biomarker Development: Using IL1RAPL1 antibodies to identify patient subgroups with specific synaptic phenotypes

  • Pharmacodynamic Markers: Measuring IL1RAPL1 localization or interaction changes to assess therapeutic responses

  • Companion Diagnostics: Developing antibody-based assays to identify patients likely to respond to specific therapies

• Therapeutic Antibody Engineering:

  • Function-Modulating Antibodies: Developing antibodies that enhance IL1RAPL1 stability or function

  • Domain-Specific Targeting: Creating antibodies that selectively promote beneficial interactions (e.g., with PTPδ) while avoiding disruption of others

  • Bispecific Antibodies: Engineering antibodies that simultaneously target IL1RAPL1 and compensatory proteins to enhance therapeutic effects

• Drug Discovery Platforms:

  • High-Throughput Screening: Using IL1RAPL1 antibodies to identify compounds that restore proper localization of mutant proteins

  • Target Validation: Confirming IL1RAPL1-associated pathways as therapeutic targets in patient-derived neurons

  • Mechanism of Action Studies: Elucidating how candidate therapeutics affect IL1RAPL1 function and localization

• Protein Replacement Strategies:

  • Antibody-Guided Delivery: Using IL1RAPL1 antibodies to direct therapeutic cargo to affected synapses

  • Stabilization of Mutant Proteins: Developing compounds that bind mutant IL1RAPL1 (like Δex6 or C31R) and prevent degradation

  • Chaperone Therapeutics: Identifying molecules that help mutant IL1RAPL1 fold properly and reach synapses

• Synaptic Function Restoration:

  • Structural Analysis: Using antibodies to define binding interfaces for small-molecule drug design

  • Pathway-Specific Intervention: Targeting downstream effectors like RhoGAP2 when IL1RAPL1 function cannot be restored

  • Compensatory Mechanism Enhancement: Identifying and boosting parallel pathways that can substitute for IL1RAPL1 function

• Targeted Delivery Systems:

  • Blood-Brain Barrier Penetrating Antibodies: Engineering IL1RAPL1 antibodies that can cross the BBB for CNS delivery

  • Nanoparticle Conjugation: Attaching therapeutic payloads to IL1RAPL1-targeting antibodies

  • Viral Vector Targeting: Directing gene therapy vectors to IL1RAPL1-expressing neurons

• Precision Medicine Applications:

  • Mutation-Specific Therapies: Developing distinct approaches for different IL1RAPL1 mutations based on antibody-revealed mechanisms

  • Combination Therapies: Identifying synergistic interventions that address multiple aspects of IL1RAPL1 dysfunction

  • Treatment Monitoring: Using antibodies to assess restoration of proper IL1RAPL1 localization and function during treatment

These approaches represent a promising frontier in intellectual disability therapeutics, potentially leading to interventions specifically tailored to correct the synaptic defects caused by IL1RAPL1 dysfunction. The detailed molecular understanding provided by antibody-based research shows that IL1RAPL1 mutations can affect protein stability, localization, and interaction with partners like PTPδ and RhoGAP2 , suggesting multiple points for therapeutic intervention.

What methodological innovations are needed to better characterize IL1RAPL1 interactions in human brain samples?

Several methodological innovations are needed to better characterize IL1RAPL1 interactions in human brain samples:

• Tissue Preparation and Preservation Innovations:

  • Rapid Fixation Protocols: Develop methods to preserve protein-protein interactions in post-mortem human tissue with minimal delay

  • Cryopreservation Techniques: Optimize cryoprotectants and freezing rates to maintain native IL1RAPL1 complexes

  • Live-to-Fixed Correlation: Establish parameters that allow extrapolation from fixed human samples to living conditions

• Single-Molecule Detection Methods:

  • Single-Molecule Pull-Down (SiMPull): Adapt for human tissue to visualize individual IL1RAPL1 complexes

  • Single-Molecule FRET: Develop protocols to measure nanoscale distances between IL1RAPL1 and binding partners in human synapses

  • Super-Resolution In Situ Techniques: Implement STORM/PALM approaches optimized for human brain tissue sections

• Spatial Multi-Omics Integration:

  • Spatial Proteomics: Map IL1RAPL1 interaction networks across different brain regions with subcellular resolution

  • Antibody-Based Proximity Labeling: Adapt BioID or APEX2 systems for use with IL1RAPL1 antibodies in human samples

  • Multiplex Imaging Mass Cytometry: Simultaneously visualize dozens of proteins in IL1RAPL1 complexes across human brain sections

• Human-Specific Antibody Development:

  • Humanized Nanobodies: Engineer smaller antibody fragments optimized for human IL1RAPL1 epitopes

  • Isoform-Specific Antibodies: Develop tools to distinguish between human IL1RAPL1 splice variants

  • Post-Translational Modification-Specific Antibodies: Create antibodies that detect specific phosphorylation or ubiquitination states

• Cross-Linking Methodologies:

  • In Situ Chemical Cross-Linking: Develop mild cross-linking protocols to stabilize transient interactions before extraction

  • Photo-Activatable Cross-Linkers: Implement spatially and temporally controlled cross-linking in human tissue

  • MS-Compatible Cross-Linking: Optimize methods for downstream mass spectrometry analysis of IL1RAPL1 complexes

• Multi-Scale Correlated Microscopy:

  • Correlative Light-Electron Microscopy (CLEM): Visualize IL1RAPL1 antibody labeling at both light and electron microscopy levels

  • Array Tomography: Serial ultrathin sections for high-resolution 3D reconstruction of IL1RAPL1 complexes

  • Expansion Microscopy Optimization: Adapt protocols for human tissue to achieve nanoscale resolution with standard microscopes

• Functional Readout Integration:

  • Patch-Seq Adaptations: Combine electrophysiology with transcriptomics and proteomics in human neurons

  • Activity-Dependent Labeling: Develop tools to selectively tag active IL1RAPL1 complexes in human tissue

  • Optogenetic Sensors: Create reporters of IL1RAPL1 interaction states for functional studies

• Human-Specific Models for Validation:

  • Patient-Derived Cerebral Organoids: Generate 3D brain models from patient iPSCs to validate findings from post-mortem tissue

  • Human Synaptosomes: Optimize isolation of intact human synapses for biochemical and imaging studies

  • Human-Mouse Chimeric Models: Develop methods to study human IL1RAPL1 function in vivo

These methodological innovations would address critical challenges in studying IL1RAPL1 in the human brain, including tissue quality issues, the complexity of human neural circuits, and species differences that limit the translation of findings from animal models. Such advances would provide unprecedented insights into how IL1RAPL1 dysfunction contributes to intellectual disability and potentially identify new therapeutic targets for intervention.

How should researchers document antibody validation data when publishing IL1RAPL1 studies?

Researchers should comprehensively document IL1RAPL1 antibody validation when publishing studies using the following structured approach:

• Antibody Specification Documentation:

  • Complete Identifiers: Include catalog number, clone designation, lot number, and RRID (Research Resource Identifier)

  • Physical Characteristics: Document host species, clonality, immunogen sequence (amino acids 564-679 for many commercial antibodies) , isotype, and any conjugates (e.g., FITC)

  • Source Information: Specify commercial vendor or custom development details with complete contact information

  • Storage Conditions: Report antibody concentration, buffer composition, and storage requirements

• Validation Methods Table:

  Validation Method	Experimental Design	Controls Used	Results	Limitations
  Western Blot	Brain lysate from x region, y μg loaded	KO tissue, overexpression	Single band at ~115 kDa	Not effective in fixed tissues
  Immunoprecipitation	Pull-down from neuronal lysate	IgG control, pre-immune serum	Enriched IL1RAPL1 verified by MS	Limited by antibody affinity
  Immunofluorescence	DIV21 neurons, 4% PFA fixation	KO neurons, peptide competition	Punctate staining at excitatory synapses	Background in glial cells
  Proximity Ligation	Co-PLA with known partners (PTPδ, RhoGAP2)	Secondary-only, non-interacting protein	Signal at synaptic junctions	Dependent on partner antibody quality

• Specificity Evidence:

  • Genetic Controls: Document testing in IL1RAPL1 knockout/knockdown tissues with quantitative signal comparison

  • Peptide Competition: Report protocol and results of pre-absorption with immunizing peptide

  • Orthogonal Methods: Compare antibody results with mRNA localization or tagged protein expression

  • Cross-Reactivity Testing: Document testing against close family members (e.g., IL1RAPL2) or potential cross-reactive proteins

• Application-Specific Validation:

  • For Imaging Applications: Include resolution limits, detection thresholds, signal-to-noise ratios

  • For Biochemical Applications: Document efficiency metrics for immunoprecipitation, sensitivity limits

  • For Proximity Assays: Report distance constraints, false positive rates

  • For Functional Studies: Describe controls showing antibody does/doesn't affect protein function

• Detailed Methodology Reporting:

  • Sample Preparation: Complete fixation protocol, permeabilization method, blocking formula

  • Antibody Application: Concentration used, diluent composition, incubation time/temperature

  • Washing Protocols: Buffer composition, number and duration of washes

  • Detection Methods: Secondary antibody details, amplification steps, imaging parameters

• Batch Variation Management:

  • Lot-to-Lot Testing: Document comparison between antibody lots if multiple lots were used

  • Standard Sample Testing: Include results from standard positive control samples across experiments

  • Calibration Curves: When applicable, include dilution series to demonstrate linear response range

• Limitations and Failure Modes:

  • Known Limitations: Document conditions where antibody performance is suboptimal

  • Failed Applications: Report attempted applications where the antibody did not perform adequately

  • Troubleshooting Guide: Provide solutions to common issues encountered

• Data Availability:

  • Raw Data Access: Provide repository links for full-resolution unprocessed images

  • Validation Data Sharing: Upload comprehensive validation data to antibody validation repositories

  • Protocol Sharing: Provide detailed protocols via protocols.io or similar platforms

Following this documentation framework ensures transparency, reproducibility, and proper interpretation of IL1RAPL1 antibody-based findings. For example, when studying IL1RAPL1 mutations like Δex6 and C31R that affect protein stability and localization , comprehensive antibody validation data allows readers to distinguish between true biological effects and technical artifacts.

How can researchers optimize antibody selection for different IL1RAPL1 research applications in neurodevelopmental disorders?

Researchers can optimize antibody selection for different IL1RAPL1 research applications in neurodevelopmental disorders by considering the following comprehensive framework:

• Application-Specific Selection Criteria:

  For Immunohistochemistry/Immunofluorescence:

  • Select antibodies validated specifically for fixed tissue applications

  • Prioritize antibodies targeting extracellular domains for surface staining

  • Consider polyclonal antibodies for increased sensitivity in tissue sections

  • For co-localization studies, choose antibodies raised in different host species than partner protein antibodies

  For Western Blotting:

  • Select antibodies validated against denatured protein

  • Consider epitope location relative to known mutations (e.g., avoid antibodies targeting exon 6 when studying Δex6 mutation)

  • Look for clean single-band detection at the expected molecular weight (~115 kDa for wild-type)

  • Choose unconjugated antibodies (conjugates like FITC are rarely beneficial for WB)

  For Co-Immunoprecipitation:

  • Select antibodies with proven affinity in native conditions

  • Choose antibodies targeting regions not involved in protein-protein interactions

  • Avoid antibodies whose epitopes overlap with binding sites for PTPδ or RhoGAP2

  • Consider antibodies validated specifically for IP applications

  For Super-Resolution Microscopy:

  • Select directly conjugated antibodies or Fab fragments for better spatial resolution

  • Choose photostable fluorophores for sustained imaging (AlexaFluor may outperform FITC)

  • Prioritize monoclonal antibodies for consistent epitope targeting

  • Consider the accessibility of epitopes in densely packed synaptic structures

• Domain-Specific Selection Strategy:

  IL1RAPL1 Domain	Research Question	Recommended Antibody Type	Considerations
  Extracellular Ig-like domains	Surface expression, PTPδ interaction	Anti-extracellular domain, non-permeabilizing conditions	Avoid antibodies that block PTPδ binding if studying interactions
  Transmembrane domain	Membrane insertion, trafficking	Antibodies to adjacent regions, permeabilizing conditions	Limited epitope availability in this hydrophobic region
  TIR domain	RhoGAP2 interaction , signaling	Anti-TIR domain antibodies	Avoid epitopes overlapping with RhoGAP2 binding site
  C-terminal tail	Post-synaptic localization	Anti-C-terminal antibodies	Useful for mutation studies as C-terminus is often preserved

• Mutation-Specific Considerations:

  • For Δex6 mutation studies: Select antibodies targeting regions outside exon 6

  • For C31R mutation research: Consider antibodies recognizing both mutant and wild-type for comparative studies

  • For truncation mutations: Choose antibodies targeting preserved regions

  • For patient-derived samples: Validate antibody reactivity with the specific mutations present

• Technical Parameter Optimization:

  Parameter	Consideration	Example
  Affinity	Higher affinity for low-abundance detection	KD < 10 nM for synaptic IL1RAPL1 detection
  Specificity	Cross-reactivity profile relevant to application	Verify no cross-reactivity with IL1RAPL2
  Sensitivity	Detection limit appropriate for expression level	LOD < 10 ng for endogenous detection
  Species reactivity	Match to experimental model	Human-mouse cross-reactivity for translational studies
  Format	Conjugates appropriate for application	FITC-conjugated for direct immunofluorescence

• Validation Quality Assessment:

  • Review published validation data for each candidate antibody

  • Prioritize antibodies validated in multiple applications and by independent laboratories

  • Consider antibodies validated specifically in neurodevelopmental disorder contexts

  • Evaluate reproducibility across different lots

• Strategic Combinations for Complex Questions:

  • Protein Dynamics Studies: Combine surface-labeling antibodies with internalization assays

  • Interaction Studies: Use differentially labeled antibodies against IL1RAPL1 and binding partners

  • Conformational Studies: Select antibodies targeting different epitopes to detect conformational changes

  • Mutation Impact Assessment: Compare multiple domain-specific antibodies to assess structural effects