MRX10 Antibody

What is MRX10 antibody and what does it recognize?

MRX10 antibody is one of several alternative designations for IL1RAPL1 antibody, which specifically recognizes the IL1RAPL1 protein (Interleukin 1 Receptor Accessory Protein-Like 1). The antibody is typically available as a mouse monoclonal format that targets human IL1RAPL1. This protein has been implicated in X-linked intellectual disability and plays a role in regulating dendrite complexity and neuronal development .

The antibody is commonly referenced by several alternative names in the literature including IL1R8, MRX10, MRX21, MRX34, OPHN4, IL1RAPL, TIGIRR-2, IL1RAPL-1, IL-1RAPL-1, and IL-1-RAPL-1 antibody. When designing experiments, researchers should be aware of these alternative designations to ensure comprehensive literature searches .

What are the validated applications for MRX10/IL1RAPL1 antibody?

The IL1RAPL1/MRX10 antibody has been validated for multiple experimental techniques through rigorous testing. Primary applications include:

• Western Blot (WB): Validated at dilutions of 1:500-1:2,000

• Immunocytochemistry (ICC): Validated at dilutions of 1:50-1:200

• Flow Cytometry (FC): Validated at dilutions of 1:100-1:200

Validation experiments have demonstrated specificity using multiple cell types including:

• HEK293 cells (both non-transfected and IL1RAPL1-hIgGFc transfected cells)

• A431 cells

• SK-Hep-1 cells

• HL-7702 cells

• HeLa cells

How should MRX10/IL1RAPL1 antibody be stored and handled for optimal performance?

For optimal performance and longevity of the MRX10/IL1RAPL1 antibody, follow these research-validated storage and handling protocols:

• Short-term storage: Store at +4°C after thawing

• Long-term storage: Aliquot and store at -20°C or -80°C

• Avoid repeated freeze/thaw cycles as this can degrade antibody performance

• The antibody is typically supplied in a liquid formulation containing 1×TBS (pH 7.4), 1% BSA, and 40% glycerol with 0.05% sodium azide as a preservative

Always centrifuge the antibody briefly before opening the vial to ensure all material is at the bottom of the tube. When making working dilutions, use fresh, cold buffer and prepare only the amount needed for immediate use to maintain antibody integrity.

How does sample preparation affect MRX10/IL1RAPL1 antibody detection sensitivity in Western blots?

Sample preparation significantly impacts the detection sensitivity of IL1RAPL1/MRX10 antibody in Western blot applications. Based on validation studies, the following methodological considerations are critical:

For cell lysate preparation, total protein extraction protocols using detergent-based lysis buffers (containing Triton X-100 or RIPA buffer) yield better results than more stringent extraction methods. Validation data shows successful detection in multiple cell lines including A431, SK-Hep-1, and HL-7702 cells .

When working with recombinant proteins, the antibody demonstrates high specificity, as evidenced by clear detection of human IL1RAPL1 recombinant protein and IL1RAPL1-hIgGFc transfected HEK293 cell lysates .

Denaturation conditions should be carefully controlled - excessive heat or strong reducing conditions may destroy the epitope recognized by this antibody. Standard SDS-PAGE sample preparation (5 minutes at 95°C in Laemmli buffer with β-mercaptoethanol) has been validated in published protocols .

For optimal results with difficult samples, researchers should consider:

• Using phosphatase and protease inhibitors in lysis buffers

• Avoiding prolonged storage of prepared samples

• Quantifying protein content and loading equal amounts across lanes

• Confirming transfer efficiency with reversible protein stains before immunoblotting

What are the critical parameters for optimizing IL1RAPL1/MRX10 antibody performance in immunocytochemistry?

Optimizing IL1RAPL1/MRX10 antibody performance in immunocytochemistry requires careful attention to several experimental parameters:

• Fixation method: Paraformaldehyde fixation (typically 4%) has been validated in ICC protocols with this antibody. Overfixation may mask epitopes, while underfixation can lead to poor morphology preservation .

• Permeabilization: 0.25% Triton X-100 in PBS has been validated for successful permeabilization with this antibody, allowing access to intracellular target proteins .

• Blocking conditions: Effective blocking minimizes non-specific binding. For IL1RAPL1 antibody, standard blocking solutions containing 1-5% BSA or normal serum from the species of the secondary antibody have proven effective.

• Antibody dilution: Validation studies recommend 1:50-1:200 dilution ranges for ICC applications . Researchers should perform titration experiments to determine optimal concentration for their specific experimental system.

• Counterstaining: Nuclear counterstaining with DAPI provides excellent contrast when visualizing cytoplasmic or membrane IL1RAPL1 localization, as demonstrated in validation studies with HeLa cells .

• Controls: Include appropriate controls for accurate interpretation:

  • Negative controls: Omission of primary antibody

  • Specificity controls: Cells known to be negative for IL1RAPL1 expression

  • Blocking peptide controls: Pre-incubation of antibody with immunizing peptide

What methodological approaches should be used when studying protein-protein interactions involving IL1RAPL1?

When investigating protein-protein interactions involving IL1RAPL1, researchers should consider several methodological approaches:

• Co-immunoprecipitation (Co-IP): This technique can be effectively performed using anti-IL1RAPL1 antibody for immunoprecipitation followed by Western blot for interacting partners. Optimal conditions include:

  • Lysis in buffers containing 150 mM NaCl, 0.5% Triton X-100, 50 mM Tris, 1.5 mM MgCl₂ (pH 7.5)

  • Addition of nuclease (e.g., 25 units/ml Benzonase) to eliminate DNA-mediated interactions

  • Inclusion of protease inhibitor cocktail

  • Conjugation of antibody to Protein A-Sepharose beads (2 μg antibody per immunoprecipitation)

• Protein stability assays: Cycloheximide chase assays can determine if IL1RAPL1 stability is affected by interacting proteins. This approach requires:

  • Treatment with 100 μg/ml cycloheximide to block protein synthesis

  • Collection of time points (0, 2, 4, 6, 8, and 10 hours)

  • Western blot analysis to track protein degradation rates

• Proximity-based methods: Techniques like proximity ligation assay (PLA) can detect protein interactions in situ with high sensitivity and specificity, requiring:

  • Primary antibodies from different species targeting IL1RAPL1 and its potential binding partner

  • Species-specific PLA probes

  • Optimal fixation and permeabilization as described for ICC applications

What are the most common causes of false negative results when using MRX10/IL1RAPL1 antibody in Western blots?

False negative results with MRX10/IL1RAPL1 antibody in Western blotting can arise from several methodological issues. A systematic troubleshooting approach should consider:

• Protein expression levels: IL1RAPL1 may be expressed at low levels in certain cell types. Validation data shows variable expression across cell lines (A431, SK-Hep-1, HL-7702) . Consider:

  • Using positive control samples (e.g., IL1RAPL1-transfected HEK293 cells)

  • Increasing protein loading (50-100 μg total protein)

  • Using enhanced chemiluminescence detection systems

• Protein extraction efficiency: IL1RAPL1 is a membrane-associated protein that may require specific extraction conditions:

  • Use detergent-based lysis buffers containing 0.5% Triton X-100

  • For difficult samples, consider stronger extraction buffers containing 1% SDS

  • Ensure complete cell lysis by sonication or mechanical disruption

• Transfer conditions: Inefficient transfer of high molecular weight proteins:

  • Use lower percentage gels (8-10% acrylamide)

  • Extend transfer time or use semi-dry transfer systems

  • Consider adding SDS (0.1%) to transfer buffer to improve high molecular weight protein transfer

• Antibody concentration: The recommended dilution range of 1:500-1:2,000 may need optimization for specific samples .

• Detection method sensitivity: Standard ECL may not be sufficient for low abundance proteins:

  • Use high-sensitivity ECL substrates

  • Consider fluorescent secondary antibodies with digital imaging

  • Increase exposure time when using film-based detection

How can researchers distinguish between specific and non-specific binding in flow cytometry applications?

Distinguishing between specific and non-specific binding in flow cytometry using MRX10/IL1RAPL1 antibody requires rigorous controls and optimization:

• Isotype controls: Use mouse IgG1 isotype control antibodies at the same concentration as the IL1RAPL1 antibody to assess background binding due to Fc receptors or non-specific interactions .

• Blocking protocols: Implement effective blocking steps to minimize non-specific binding:

  • Pre-incubate cells with 5-10% normal serum from the secondary antibody species

  • Add 1% BSA to staining buffer

  • Consider Fc receptor blocking reagents when working with immune cells

• Titration experiments: Determine the optimal antibody concentration by testing serial dilutions (1:50, 1:100, 1:200, 1:400) to identify the concentration that maximizes specific signal while minimizing background .

• Negative controls: Include cell populations known to be negative for IL1RAPL1 expression. Validation data has been established using HeLa cells, which can serve as a reference population .

• Competitive binding assays: Pre-incubate the antibody with excess immunizing peptide to block specific binding sites, which should eliminate true positive signals while non-specific binding would remain.

• Signal-to-noise ratio analysis: Calculate the staining index (mean fluorescence intensity of positive population divided by twice the standard deviation of the negative population) to quantitatively assess staining quality.

How can MRX10/IL1RAPL1 antibody be used to study neurodevelopmental disorders?

MRX10/IL1RAPL1 antibody serves as a valuable tool for investigating neurodevelopmental disorders, particularly X-linked intellectual disability, where IL1RAPL1 mutations have been implicated:

• Expression analysis in patient-derived samples: IL1RAPL1 antibody can be used to assess protein expression levels in:

  • Patient-derived lymphoblasts or fibroblasts

  • Induced pluripotent stem cell (iPSC)-derived neurons

  • Post-mortem brain tissue samples

• Dendritic complexity assessment: IL1RAPL1 has been demonstrated to regulate dendrite complexity in neurons . Researchers can employ:

  • ICC with IL1RAPL1 antibody (1:50-1:200 dilution) combined with neuronal markers

  • Quantitative morphometric analysis of dendritic arbors in IL1RAPL1-deficient models

  • Co-localization studies with synaptic markers to assess synaptic distribution

• Functional studies: The antibody can be used to:

  • Validate knockdown or knockout models via Western blot (1:500-1:2,000 dilution)

  • Confirm overexpression in rescue experiments

  • Isolate IL1RAPL1-containing protein complexes through immunoprecipitation

• Biomarker development: Flow cytometry applications (1:100-1:200 dilution) could potentially identify altered IL1RAPL1 expression patterns associated with specific clinical phenotypes .

What experimental considerations should be made when using MRX10/IL1RAPL1 antibody in cross-species research?

When employing MRX10/IL1RAPL1 antibody across different species, researchers should consider several critical experimental factors:

• Species reactivity: The antibody is primarily validated for human IL1RAPL1 detection . For cross-species applications:

  • Perform sequence alignment analysis of the immunogen region across target species

  • Validate antibody reactivity using positive control samples from each species

  • Consider testing multiple antibodies targeting different epitopes for confirmation

• Epitope conservation: IL1RAPL1 contains conserved domains across species, but researchers should:

  • Check manufacturer specifications for validated species reactivity

  • Perform preliminary Western blot validation when working with non-validated species

  • Consider epitope mapping for precise cross-reactivity assessment

• Alternative detection methods: When antibody cross-reactivity is limited:

  • Complement antibody-based detection with mRNA expression analysis

  • Use species-specific antibodies when available

  • Consider tagging approaches (e.g., FLAG, HA) in experimental models

• Quantitative considerations: When comparing IL1RAPL1 levels across species:

  • Normalize to appropriate housekeeping proteins for each species

  • Use recombinant protein standards for absolute quantification

  • Validate antibody affinity differences that might affect quantitative comparisons

How can researchers employ computational approaches to predict antibody specificity when using MRX10/IL1RAPL1 antibody?

Integrating computational approaches with experimental validation can enhance specificity prediction when working with MRX10/IL1RAPL1 antibody:

• Epitope prediction and cross-reactivity analysis:

  • Identify the IL1RAPL1 epitope recognized by the antibody through manufacturer specifications

  • Use BLAST or similar tools to identify proteins with sequence similarity

  • Perform structural modeling to assess epitope accessibility in native protein conformations

  • Analyze post-translational modifications that might affect antibody recognition

• Machine learning approaches:

  • Utilize biophysics-informed modeling to predict antibody-antigen interactions

  • Apply machine learning algorithms trained on antibody-epitope datasets to predict binding affinities

  • Integrate experimental validation data to refine computational models

• Off-target binding prediction:

  • Analyze proteome-wide potential cross-reactive targets through sequence and structural homology

  • Consider both linear and conformational epitopes in prediction algorithms

  • Validate predictions through experimental approaches like immunoprecipitation followed by mass spectrometry

• Data integration strategies:

  • Combine computational predictions with experimental validation data

  • Implement iterative approaches that refine models based on experimental outcomes

  • Develop confidence scores for binding predictions based on multiple parameters

What are the advantages and limitations of monoclonal versus polyclonal antibodies for IL1RAPL1 detection?

Best practice recommendation: For critical quantitative experiments, use the monoclonal IL1RAPL1/MRX10 antibody with validated positive controls. For exploratory work or when epitope accessibility may be an issue, consider complementary approaches with polyclonal antibodies or alternative detection methods.

What methodological controls should be implemented when studying low-abundance IL1RAPL1 expression?

When investigating low-abundance IL1RAPL1 expression, implementing rigorous methodological controls is essential for reliable results:

• Positive expression controls:

  • IL1RAPL1-transfected cell lines (e.g., IL1RAPL1-hIgGFc transfected HEK293)

  • Tissues with known IL1RAPL1 expression (e.g., neural tissues)

  • Recombinant IL1RAPL1 protein standards for quantitative analysis

• Negative expression controls:

  • Non-transfected parental cell lines

  • Tissues or cells with confirmed absence of IL1RAPL1 expression

  • siRNA/shRNA knockdown controls to confirm antibody specificity

• Technical controls for Western blot applications:

  • Loading controls (e.g., β-actin, GAPDH) to normalize protein amounts

  • Molecular weight markers to confirm target band size

  • Serial dilution of positive control samples to establish detection limits

  • Enrichment strategies (e.g., immunoprecipitation) before Western blot to concentrate target protein

• Flow cytometry methodological controls:

  • Isotype control (Mouse IgG1) at matching concentration

  • Unstained cells to establish autofluorescence baseline

  • Single-color controls for compensation when using multiple fluorophores

  • Titration experiments to determine optimal antibody concentration

• Immunocytochemistry controls:

  • Peptide competition assays to confirm binding specificity

  • Secondary antibody-only controls to assess background

  • Counterstaining with established markers (e.g., actin filaments) for reference