IL1RAPL1 Antibody, HRP conjugated

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

IL1RAPL1 is a member of the interleukin 1 receptor family that shares structural similarities with interleukin 1 accessory proteins. It is most closely related to IL1RAPL2 and is located on chromosome X in a region associated with X-linked non-syndromic mental retardation. The significance of IL1RAPL1 in neuroscience research stems from its high expression levels in post-natal brain structures involved in the hippocampal memory system and its role in excitatory synapse formation. Deletions and mutations in IL1RAPL1 have been found in patients with cognitive impairments ranging from non-syndromic X-linked mental retardation to autism, suggesting a specialized role in the physiological processes underlying memory and learning abilities .

IL1RAPL1 regulates the synaptic localization of PSD-95 through control of c-Jun N-terminal kinase activity and PSD-95 phosphorylation. Additionally, the IgG-like extracellular domains of IL1RAPL1 induce excitatory pre-synapse formation by interacting with protein tyrosine phosphatase delta (PTPδ), while its TIR domains interact with RhoGAP2 at excitatory post-synaptic density to facilitate dendritic spine formation .

What are the key applications for HRP-conjugated IL1RAPL1 antibodies?

HRP-conjugated IL1RAPL1 antibodies are particularly valuable in ELISA applications where direct detection without secondary antibodies is beneficial. The HRP (horseradish peroxidase) conjugation enables direct detection through enzymatic reaction with substrate, resulting in colorimetric, chemiluminescent, or fluorescent signals depending on the substrate used . While the primary application of commercially available HRP-conjugated IL1RAPL1 antibodies is ELISA, these antibodies can also potentially be used in immunohistochemistry and western blotting with appropriate optimization.

The specific binding to amino acids 564-679 of human IL1RAPL1 makes these antibodies useful for detecting the protein in complex biological samples like cell lysates, tissue homogenates, or brain section analyses. This region is part of the intracellular domain of IL1RAPL1, which is critical for its interaction with downstream signaling molecules .

How should I store and handle HRP-conjugated IL1RAPL1 antibodies?

Proper storage and handling of HRP-conjugated antibodies is critical for maintaining their activity. HRP-conjugated IL1RAPL1 antibodies should be stored at -20°C for long-term storage. The typical formulation includes glycerol (often around 50%) and a neutral pH buffer with preservatives to maintain stability .

To preserve enzymatic activity, avoid repeated freeze-thaw cycles by aliquoting the antibody upon first thaw. When handling the antibody, keep it on ice when in use and return to storage promptly. Exposure to high temperatures, extreme pH conditions, and certain chemicals (particularly sodium azide, which can inhibit HRP activity) should be avoided. Additionally, protect HRP-conjugated antibodies from prolonged exposure to light, as this can gradually reduce the enzymatic activity of the conjugate .

What is the recommended protocol for using HRP-conjugated IL1RAPL1 antibodies in ELISA?

For optimal results with HRP-conjugated IL1RAPL1 antibodies in ELISA, follow this methodological approach:

• Coating: Coat microtiter plate wells with the antigen of interest (purified protein, cell lysate containing IL1RAPL1, or capture antibody for sandwich ELISA) in coating buffer (typically carbonate/bicarbonate buffer, pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with a blocking buffer (typically 1-5% BSA or non-fat dry milk in PBS or TBS) for 1-2 hours at room temperature.

• Antibody Dilution: Dilute the HRP-conjugated IL1RAPL1 antibody in blocking buffer or antibody dilution buffer. The recommended dilution range is usually 1:500-1:2000, but optimal dilution should be determined empirically for each experimental setup .

• Incubation: Add diluted antibody to wells and incubate for 1-2 hours at room temperature or overnight at 4°C with gentle shaking.

• Washing: Wash wells thoroughly with washing buffer (PBS or TBS with 0.05-0.1% Tween-20) at least 4-5 times.

• Detection: Add appropriate HRP substrate (TMB, ABTS, or chemiluminescent substrate) and allow color development.

• Stopping Reaction: Stop the reaction with stopping solution (e.g., 2N H₂SO₄ for TMB) if using a colorimetric substrate.

• Measurement: Measure absorbance or luminescence using a plate reader at the appropriate wavelength for the substrate used.

Include appropriate positive and negative controls to validate results and establish a standard curve if quantification is needed .

How can I optimize the dilution ratio for HRP-conjugated IL1RAPL1 antibodies?

Optimizing the dilution ratio for HRP-conjugated IL1RAPL1 antibodies is essential for achieving the best signal-to-noise ratio. A systematic approach includes:

• Titration Series: Perform a checkerboard titration using serial dilutions of both antigen (when applicable) and antibody. Start with the manufacturer's recommended dilution (typically 1:500-1:2000 for HRP-conjugated IL1RAPL1 antibodies) and test at least 3-4 dilutions above and below this range .

• Signal-to-Noise Assessment: For each dilution, calculate the signal-to-noise ratio by dividing the signal from positive samples by the background signal from negative controls. The optimal dilution provides the highest signal-to-noise ratio, not necessarily the strongest absolute signal.

• Incubation Conditions: Evaluate different incubation times and temperatures. Sometimes, longer incubation at 4°C may yield better results than shorter incubation at room temperature.

• Substrate Exposure Time: For HRP detection, optimize the substrate exposure time to avoid signal saturation or excessive background development.

A systematic optimization table should be created to document results:

Remember that each new lot of antibody may require re-optimization, as conjugation efficiency can vary between lots .

How can I verify the specificity of IL1RAPL1 antibodies in my experimental system?

Verifying antibody specificity is crucial for reliable research results. For IL1RAPL1 antibodies, consider these methodological approaches:

• Positive and Negative Controls: Include tissues or cell lines known to express or lack IL1RAPL1. For IL1RAPL1, neuronal cell lines or tissues from hippocampal regions would serve as positive controls, while non-neural tissues might serve as negative controls .

• Western Blot Analysis: Confirm that the antibody detects a band of the expected molecular weight. For IL1RAPL1, the calculated molecular weight is approximately 79 kDa, although the observed molecular weight is often around 90 kDa due to post-translational modifications .

• Knockdown/Knockout Validation: If possible, test the antibody on samples where IL1RAPL1 has been knocked down (siRNA) or knocked out (CRISPR-Cas9). A specific antibody should show reduced or absent signal in these samples.

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide before application to your samples. Specific binding should be blocked by this competition, resulting in signal reduction.

• Cross-Reactivity Testing: Verify that the antibody does not cross-react with related proteins like IL1RAPL2 or other IL-1 receptor family members. Commercial IL1RAPL1 antibodies are often tested against these potential cross-reactants .

• Multiple Antibody Comparison: When possible, compare results using antibodies targeting different epitopes of IL1RAPL1 to confirm consistent detection patterns.

Document all validation experiments thoroughly to ensure reproducibility and reliability of subsequent studies .

How can IL1RAPL1 antibodies be used to study synapse formation in neuronal cultures?

IL1RAPL1 antibodies can be powerful tools for investigating synapse formation, given IL1RAPL1's role in excitatory synapse development. A comprehensive methodological approach includes:

• Immunocytochemistry for Synaptic Localization: Use IL1RAPL1 antibodies in conjunction with markers for pre-synaptic (e.g., synapsin, synaptophysin) and post-synaptic (e.g., PSD-95, homer) structures to visualize co-localization at synapses. This can reveal how IL1RAPL1 distribution changes during synapse formation and maturation .

• Quantification of Synaptic Density: Employ IL1RAPL1 antibodies to quantify changes in synaptic density following genetic manipulation (overexpression or knockdown of IL1RAPL1) or pharmacological treatment. Co-staining with IL1RAPL1 antibodies and synaptic markers allows for automated image analysis to measure synaptic puncta density and size .

• Live Imaging of Synapse Dynamics: For live imaging studies, surface-labeling with IL1RAPL1 antibodies (non-HRP conjugated) followed by fluorescent secondary antibodies can track the dynamics of IL1RAPL1 during synapse formation and remodeling. Time-lapse imaging can reveal the temporal relationship between IL1RAPL1 recruitment and pre-synaptic differentiation.

• Protein-Protein Interaction Studies: IL1RAPL1 antibodies can be used in co-immunoprecipitation experiments to isolate and identify protein complexes involved in synapse formation. This approach has revealed that IL1RAPL1 interacts with PTPδ through its extracellular domain and RhoGAP2 through its intracellular TIR domain .

• Functional Assays: Combined with electrophysiological recordings, IL1RAPL1 antibody staining can correlate structural changes in synapses with functional alterations in synaptic transmission. Studies have shown that IL1RAPL1 affects miniature excitatory postsynaptic current (mEPSC) frequency but not amplitude, suggesting a role in pre-synaptic vesicle release or synapse number rather than post-synaptic receptor content .

These approaches have revealed that IL1RAPL1 induces pre-synaptic differentiation through its interaction with PTPδ and promotes dendritic spine formation through both its extracellular and intracellular domains .

What are the key considerations when using IL1RAPL1 antibodies to study protein-protein interactions?

When investigating protein-protein interactions involving IL1RAPL1, several methodological considerations are critical:

• Epitope Accessibility: The epitope recognized by your IL1RAPL1 antibody may be masked when the protein is engaged in certain interactions. For instance, antibodies targeting amino acids 564-679 may have limited access if this region is involved in binding to RhoGAP2 or other partners. Consider using multiple antibodies recognizing different epitopes of IL1RAPL1 .

• Choice of Lysis Conditions: The buffer composition can significantly affect the preservation of protein-protein interactions. For membrane-associated proteins like IL1RAPL1, which is a single-pass type I membrane protein, mild detergents (0.5-1% NP-40, 0.5% Triton X-100) are preferable to stronger denaturing agents that might disrupt interactions .

• Cross-linking Approaches: For transient or weak interactions, consider using chemical cross-linkers before immunoprecipitation. This can "freeze" interactions that might otherwise be lost during purification steps.

• Co-immunoprecipitation Controls: Include appropriate controls when performing co-IP with IL1RAPL1 antibodies:

  • IgG control to assess non-specific binding

  • Lysates from cells not expressing IL1RAPL1 or with IL1RAPL1 knocked down

  • Competition with immunizing peptide to confirm specificity

• Directionality of Interaction Testing: Test the interaction bidirectionally by immunoprecipitating with antibodies against both IL1RAPL1 and its suspected binding partners (e.g., PTPδ or RhoGAP2) to confirm the interaction .

• Domain-Specific Interactions: Use domain deletion constructs (like IL1RAPL1ΔN, which lacks the extracellular domain) to map interaction domains. Research has shown that full-length IL1RAPL1, but not IL1RAPL1ΔN, immunoprecipitates PTPδ, confirming the extracellular domain as the interaction site .

These methodological considerations are essential for accurately characterizing IL1RAPL1's interactome and understanding its role in synapse formation and function .

Why might the observed molecular weight of IL1RAPL1 differ from the predicted size in Western blot?

The discrepancy between predicted and observed molecular weights of IL1RAPL1 in Western blot analysis is a common issue that researchers encounter. The calculated molecular weight of IL1RAPL1 is approximately 79 kDa, while the observed molecular weight is often around 90 kDa . Several methodological factors can explain this difference:

• Post-translational Modifications: IL1RAPL1 undergoes various post-translational modifications, particularly glycosylation, which can significantly increase its apparent molecular weight. The extracellular domain contains potential N-glycosylation sites that can add substantial mass to the protein .

• Protein Conformation: Even under denaturing conditions, some proteins may not fully linearize, causing them to migrate differently than expected based on their amino acid sequence alone.

• Protein-Specific Properties: The amino acid composition of IL1RAPL1, particularly charged residues and hydrophobic regions associated with its transmembrane domain, can affect its migration pattern in SDS-PAGE.

• Technical Factors:

  • Gel percentage: Lower percentage gels may not resolve higher molecular weight proteins accurately

  • Buffer conditions: The pH and ionic strength of running buffer can affect migration

  • Voltage and run time: Higher voltage can cause band distortion

To address these issues methodologically:

• Use protein ladders with closely spaced markers in the 70-100 kDa range for more accurate sizing

• Include positive control samples with validated IL1RAPL1 expression

• Consider using gradient gels (4-12% or 4-20%) for better resolution of proteins across a broad molecular weight range

• For confirmatory analysis, perform immunoprecipitation followed by mass spectrometry to precisely identify the protein

How can background signal be reduced when using HRP-conjugated IL1RAPL1 antibodies?

Excessive background signal can significantly impair the quality and interpretability of results when using HRP-conjugated IL1RAPL1 antibodies. Several methodological approaches can help minimize background:

• Optimize Blocking Conditions:

  • Test different blocking agents (BSA, casein, non-fat dry milk, commercial blocking buffers)

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Consider adding 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Antibody Dilution Optimization:

  • Use a higher dilution of the HRP-conjugated antibody (start with 1:2000 and increase if needed)

  • Dilute antibody in blocking buffer containing 0.05-0.1% Tween-20

  • Consider adding 1-5% of the blocking agent to the antibody dilution buffer

• Washing Protocol Enhancement:

  • Increase washing frequency (6-8 washes instead of standard 3-5)

  • Extend wash duration (5-10 minutes per wash)

  • Use higher concentrations of Tween-20 in wash buffer (0.1-0.2%)

  • Consider adding low salt (150-300 mM NaCl) to wash buffer to reduce ionic interactions

• Substrate Considerations:

  • Use substrate with appropriate sensitivity (TMB for colorimetric, enhanced chemiluminescent substrates for Western blots)

  • Reduce substrate incubation time if background develops quickly

  • For ECL detection, dilute substrate components if signal is overwhelming

• Sample Preparation:

  • Pre-clear samples with Protein G beads or irrelevant antibodies of the same species

  • Use freshly prepared samples when possible

  • Ensure complete removal of SDS or other detergents that might interfere with antibody binding

A systematic optimization approach documenting these variables will help identify the optimal conditions for your specific experimental system .

How should contradictory results using different IL1RAPL1 antibodies be interpreted?

Contradictory results when using different IL1RAPL1 antibodies are not uncommon and require careful methodological analysis and interpretation:

• Epitope Differences: Different antibodies recognize distinct epitopes on IL1RAPL1. For example, antibodies targeting the extracellular domain (AA 19-357) versus those targeting intracellular regions (AA 564-679) may yield different results depending on protein conformation, processing, or interactions .

• Domain-Specific Functionality: IL1RAPL1 has functionally distinct domains - the extracellular domain mediates interaction with PTPδ for pre-synaptic differentiation, while both extracellular and intracellular domains are required for dendritic spine formation. Antibodies targeting different domains may therefore reveal different aspects of IL1RAPL1 function .

• Experimental Context Considerations:

  • Tissue/cell specificity: IL1RAPL1 expression and processing may vary between tissues

  • Developmental stage: IL1RAPL1 expression is high in post-natal brain structures

  • Experimental conditions: Fixation methods, detergents, or buffer compositions may differentially affect epitope accessibility

• Methodological Approach to Reconciling Differences:

  • Perform side-by-side comparison using identical samples and protocols

  • Use genetic validation (siRNA knockdown, CRISPR knockout) to confirm specificity

  • Cross-validate with non-antibody methods (RNA analysis, mass spectrometry)

  • Consider domain-specific functions when interpreting results

• Documentation and Reporting:

  • Clearly report which antibody was used (including catalog number, lot, and epitope)

  • Describe detailed methods including fixation, permeabilization, and blocking conditions

  • Report seemingly contradictory results rather than selectively reporting only "expected" outcomes

Understanding these considerations helps interpret apparent contradictions as potentially valuable insights into protein structure, processing, or context-dependent functions rather than experimental failures .

What methodologies can be employed to study IL1RAPL1's role in intellectual disability using antibody-based approaches?

Investigating IL1RAPL1's role in intellectual disability requires sophisticated methodological approaches combining antibody-based detection with functional assays:

• Patient-Derived Cell Studies:

  • Use IL1RAPL1 antibodies to compare protein expression and localization in neural cells derived from patients with IL1RAPL1 mutations versus controls

  • Analyze co-localization with synaptic markers (PSD-95, synaptophysin) to assess synaptic targeting

  • Quantify dendritic spine morphology and density using immunofluorescence with IL1RAPL1 and actin/spine markers

• Rescue Experiments:

  • In cells with IL1RAPL1 mutations, reintroduce wild-type or mutant IL1RAPL1 and use antibodies to confirm expression

  • Assess whether synaptic defects are rescued by wild-type but not mutant proteins

  • Use domain-specific constructs (extracellular or intracellular) to determine which domains are necessary for functional rescue

• Circuit-Level Analysis:

  • Use IL1RAPL1 antibodies in brain sections from animal models to map expression in specific neural circuits

  • Correlate IL1RAPL1 expression with electrophysiological measures of circuit function

  • Assess how IL1RAPL1 mutations affect circuit development and function through combined immunohistochemistry and electrophysiology

• Molecular Pathway Analysis:

  • Use phospho-specific antibodies to assess activation of downstream signaling (JNK pathway)

  • Perform co-immunoprecipitation with IL1RAPL1 antibodies followed by mass spectrometry to identify novel interacting partners

  • Study how mutations affect binding to known partners (PTPδ, RhoGAP2) using co-IP and proximity ligation assays

These methodologies have revealed that IL1RAPL1 critically regulates excitatory synapse formation through both trans-synaptic interactions (via PTPδ) and post-synaptic signaling (via RhoGAP2), providing a molecular framework for understanding how its disruption leads to intellectual disability .

How can I optimize immunohistochemistry protocols for detecting IL1RAPL1 in brain tissue sections?

Optimizing immunohistochemistry (IHC) for IL1RAPL1 detection in brain tissue requires careful consideration of sample preparation and detection methods:

• Tissue Fixation Optimization:

  • Test different fixatives: 4% paraformaldehyde (most common), methanol, or acetone

  • Vary fixation duration (2-24 hours) and temperature (4°C vs. room temperature)

  • For some epitopes, light fixation (1-2 hours) may preserve antigenicity better

  • Post-fixation storage in cryoprotectant rather than continued storage in fixative

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval: Test citrate buffer (pH 6.0) vs. Tris-EDTA (pH 9.0)

  • Enzymatic retrieval: Proteinase K, trypsin, or pepsin at different concentrations

  • Optimize retrieval duration (10-30 minutes) and temperature (80-95°C)

• Permeabilization and Blocking Strategy:

  • For membrane proteins like IL1RAPL1, use mild detergents (0.1-0.3% Triton X-100)

  • Extended blocking (2+ hours) with 5-10% normal serum from the species of secondary antibody

  • Add 0.1-0.3% Triton X-100 to blocking solution for improved penetration

• Primary Antibody Incubation Parameters:

  • Extended incubation (overnight at 4°C to 48 hours) for improved penetration

  • Optimize antibody dilution specifically for IHC (1:100-1:500 for most unconjugated antibodies)

  • For HRP-conjugated antibodies, generally use a more dilute solution than for ELISA

  • Consider addition of 0.1% BSA and 0.05% sodium azide to prevent proteolytic degradation

• Detection System Selection:

  • For HRP-conjugated antibodies: DAB, AEC, or tyramine signal amplification

  • For fluorescent detection: TSA systems can amplify signal from HRP-conjugated antibodies

  • Consider the inherent autofluorescence of brain tissue when selecting fluorophores

• Controls and Validation:

  • Include positive control tissues (hippocampus for IL1RAPL1)

  • Run parallel negative controls (secondary antibody only, isotype control)

  • Verify specificity with peptide competition or tissues from knockout models