LRRN3 Antibody

What is LRRN3 and what is its significance in research?

LRRN3, also known as leucine-rich repeat neuronal protein 3 (NLRR-3), is a membrane protein belonging to the leucine-rich repeat superfamily. The protein is approximately 79.4 kilodaltons in mass and is also referred to by several synonyms including FIGLER5, NLRR3, and fibronectin type III, immunoglobulin and leucine rich repeat domains 5 . LRRN3 contains leucine-rich repeat domains that typically mediate protein-protein interactions, suggesting its potential role in cellular signaling and neuronal development. Current research focuses on understanding its functions in neuronal systems, potential involvement in developmental processes, and possible implications in neurological disorders.

What types of LRRN3 antibodies are available for research applications?

Research-grade LRRN3 antibodies are available in several formats that vary based on the host species, clonality, target epitope region, and conjugation status:

Both polyclonal and monoclonal antibodies against different epitopes of LRRN3 are available, with rabbit being the most common host species for polyclonals . Most antibodies target either the N-terminal or C-terminal regions of the protein, while some are designed against internal sequences. The choice between these depends on the specific experimental requirements and the structural accessibility of the epitope in your particular application.

How do researchers determine cross-species reactivity of LRRN3 antibodies?

Cross-reactivity determination for LRRN3 antibodies typically involves:

• Sequence homology analysis: Comparing the amino acid sequence of the immunogen region across different species to predict potential cross-reactivity.

• Experimental validation: Testing the antibody against samples from different species using applications like Western blot or immunohistochemistry.

• Literature and database consultation: Reviewing published data and supplier information regarding validated species reactivity.

Many commercially available LRRN3 antibodies have demonstrated reactivity across human, mouse, and rat species, with some showing broader cross-reactivity to rabbit, dog, guinea pig, horse, and pig samples . When selecting an antibody for multi-species studies, prioritize those with experimentally validated cross-reactivity rather than relying solely on predicted reactivity based on sequence homology.

What are the optimal sample preparation methods for detecting LRRN3 in different applications?

Sample preparation varies by application and tissue/cell type:

For Western Blot analysis:

• Cell lysates: Use RIPA buffer supplemented with protease inhibitors

• Tissue samples: Homogenize in appropriate buffer (e.g., RIPA) with protease inhibitors

• Sample loading: 20-40 μg of total protein per lane is typically sufficient

• Denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer containing a reducing agent

For Immunohistochemistry:

• Fixation: 4% paraformaldehyde is commonly used

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is often effective

• Blocking: 5-10% normal serum (matching secondary antibody host) with 0.1-0.3% Triton X-100

• Antibody dilution: Typically 1:100 to 1:500 (optimize for each specific antibody)

For Immunofluorescence:

• Cell preparation: Grow cells on coated coverslips, fix with 4% paraformaldehyde

• Permeabilization: 0.1-0.3% Triton X-100 in PBS for 10 minutes

• Blocking: 1-5% BSA or 5-10% normal serum in PBS with 0.1% Tween-20

These methods should be optimized based on the specific LRRN3 antibody being used and the biological system under investigation .

How should researchers optimize Western blot protocols specifically for LRRN3 detection?

For optimal LRRN3 detection by Western blot, consider the following protocol modifications:

• Gel selection: Use 8-10% polyacrylamide gels to properly resolve the ~79.4 kDa LRRN3 protein

• Transfer conditions:

  • Semi-dry transfer: 15V for 30-45 minutes

  • Wet transfer: 100V for 60-90 minutes or 30V overnight at 4°C

  • PVDF membranes often produce better results than nitrocellulose for this protein

• Blocking optimization:

  • Test both 5% BSA and 5% non-fat dry milk in TBST

  • Block for 1 hour at room temperature or overnight at 4°C

• Antibody dilution and incubation:

  • Primary antibody: Typically 1:500 to 1:1000 dilution

  • Incubation: Overnight at 4°C with gentle rocking

  • Secondary antibody: 1:2000 to 1:5000 dilution for 1 hour at room temperature

• Detection system:

  • Enhanced chemiluminescence (ECL) is sufficient for most applications

  • For low expression levels, consider using more sensitive detection systems

Researchers should always conduct preliminary titration experiments to determine the optimal antibody concentration for their specific sample type and detection system .

What controls should be included when using LRRN3 antibodies in experimental workflows?

Rigorous experimental design requires appropriate controls:

Positive controls:

• Cell lines with known LRRN3 expression (neuronal cell lines are often suitable)

• Tissues with documented LRRN3 expression (brain tissue sections)

• Recombinant LRRN3 protein (particularly useful for Western blot)

Negative controls:

• Cell lines with confirmed low/no LRRN3 expression

• Tissues where LRRN3 is not expressed

• LRRN3 knockout/knockdown samples (ideal but not always available)

Technical controls:

• Primary antibody omission control

• Isotype control (especially for flow cytometry and IHC)

• Blocking peptide competition assay to confirm specificity

• Secondary antibody-only control to assess non-specific binding

For advanced studies, including siRNA knockdown or CRISPR knockout samples as controls provides the most stringent validation of antibody specificity.

What are common challenges when using LRRN3 antibodies in Western blotting and how can they be resolved?

For LRRN3 specifically, researchers should be aware that the protein runs at approximately 79.4 kDa, but post-translational modifications (particularly glycosylation) may result in higher apparent molecular weights. Additionally, some antibodies may detect specific splice variants, resulting in bands of different sizes .

Why might an LRRN3 antibody work in one application but fail in another?

This common phenomenon occurs for several reasons:

• Epitope accessibility: The three-dimensional conformation of LRRN3 differs between applications. In Western blot, proteins are denatured, exposing all epitopes, while in IHC or IF, the protein maintains native conformation, potentially obscuring certain epitopes.

• Fixation effects: Some fixatives used in IHC/IF may alter or mask the epitope recognized by the antibody.

• Application-specific sensitivity threshold: The detection limit varies between applications, with Western blot typically being more sensitive than IHC.

• Buffer incompatibility: Certain antibodies perform optimally in specific buffer conditions that differ between applications.

When an antibody performs inconsistently across applications, consider:

• Using alternative antibodies targeting different epitopes

• Modifying fixation protocols for IHC/IF

• Adjusting antigen retrieval methods

• Testing different blocking reagents specific to each application

How can researchers validate the specificity of their LRRN3 antibody?

Comprehensive antibody validation includes:

• Western blot analysis:

  • Confirm single band at expected molecular weight (~79.4 kDa)

  • Compare against recombinant LRRN3 protein standard

  • Test in LRRN3 knockdown/knockout samples

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Loss of signal confirms specificity

• Orthogonal validation:

  • Compare protein detection with mRNA expression data

  • Use multiple antibodies targeting different epitopes

• Mass spectrometry validation:

  • Immunoprecipitate LRRN3 using the antibody

  • Confirm protein identity by mass spectrometry

• Genetic validation:

  • Test in CRISPR/Cas9 knockout or siRNA knockdown systems

  • Signal should be reduced or absent compared to wildtype

This multi-faceted approach provides robust confirmation of antibody specificity and helps distinguish true signal from artifacts .

How can LRRN3 antibodies be utilized in protein-protein interaction studies?

LRRN3 antibodies can be powerful tools for investigating protein interactions through several approaches:

• Co-immunoprecipitation (Co-IP):

  • Use LRRN3 antibodies immobilized on protein A/G beads to pull down LRRN3 and associated proteins

  • Western blot analysis with antibodies against suspected interaction partners

  • Protocol optimization: Use mild lysis buffers to preserve protein-protein interactions

• Proximity Ligation Assay (PLA):

  • Detect protein interactions in situ with spatial resolution

  • Requires antibodies against LRRN3 and potential interaction partner from different host species

  • Results in fluorescent spots only where proteins are in close proximity (<40 nm)

• Immunofluorescence co-localization:

  • Use LRRN3 antibodies in combination with antibodies against potential interaction partners

  • Quantify co-localization using specialized software (e.g., JACoP plugin for ImageJ)

• Pull-down assays with tagged LRRN3:

  • Express tagged LRRN3 (His, GST, etc.)

  • Use LRRN3 antibodies to confirm expression and pull-down efficiency

  • Identify novel interaction partners by mass spectrometry

When designing these experiments, consider using site-specific LRRN3 antibodies (N-terminal vs. C-terminal) to investigate whether particular domains are involved in specific protein interactions.

What approaches can be used to study LRRN3 expression patterns across different tissues or disease states?

Comprehensive analysis of LRRN3 expression patterns requires multi-modal approaches:

• Tissue microarray (TMA) analysis:

  • Simultaneous IHC analysis of LRRN3 across multiple tissue samples

  • Quantification using digital pathology tools

  • Statistical comparison between normal and disease tissue

• Multiplexed immunofluorescence:

  • Co-staining LRRN3 with cell type-specific markers

  • Enables identification of specific cell populations expressing LRRN3

  • Use multispectral imaging systems for optimal separation of fluorophores

• Single-cell analysis:

  • Flow cytometry with permeabilization for intracellular LRRN3 detection

  • Mass cytometry (CyTOF) for high-dimensional analysis

  • Correlation with single-cell transcriptomics data

• Quantitative Western blot analysis:

  • Tissue lysate panel screening

  • Normalization to housekeeping proteins

  • Densitometric analysis for comparative expression levels

How might LRRN3 antibodies be employed in developmental neurobiology research?

LRRN3's potential role in neuronal development makes it particularly relevant for developmental neurobiology studies:

• Temporal expression analysis:

  • Western blot and IHC analysis across developmental timepoints

  • Correlation with key developmental milestones

  • Comparison between different brain regions

• Spatial expression mapping:

  • Immunohistochemistry on tissue sections at different developmental stages

  • Co-staining with markers of neurogenesis, migration, and differentiation

  • 3D reconstruction of expression patterns

• Functional studies in neuronal cultures:

  • Antibody-mediated blocking of LRRN3 function

  • Analysis of effects on neurite outgrowth, synaptogenesis, or electrophysiological properties

  • Correlation with changes in downstream signaling pathways

• In vivo developmental studies:

  • LRRN3 immunostaining in animal models at different developmental stages

  • Correlation with behavioral or functional outcomes

  • Potential use in disease models with developmental origins

When designing developmental studies, consider using antibodies validated for the specific species model system and conduct preliminary time-course experiments to identify critical windows of LRRN3 expression or function .

How can researchers optimize multiplexed imaging protocols that include LRRN3 antibodies?

Multiplexed imaging with LRRN3 antibodies requires careful planning:

• Antibody panel design:

  • Select antibodies from different host species to avoid cross-reactivity

  • Test for spectral overlap and optimize fluorophore selection

  • Consider using directly conjugated primary antibodies when possible

• Sequential staining protocols:

  • When antibodies from the same species must be used:

    • Apply first primary and secondary antibodies

    • Block with excess unconjugated Fab fragments

    • Apply subsequent antibody pairs

• Signal amplification strategies:

  • Tyramide signal amplification (TSA) for low-abundance targets

  • Quantum dots for improved signal stability and multiplexing

• Advanced imaging platforms:

  • Confocal microscopy with spectral unmixing

  • Multi-epitope ligand cartography (MELC)

  • Imaging mass cytometry for highly multiplexed analysis

• Analysis considerations:

  • Automated image segmentation for quantification

  • Machine learning approaches for pattern recognition

  • Spatial statistics for analyzing co-localization

The specific properties of your LRRN3 antibody, including sensitivity, background, and epitope accessibility, will influence the optimal multiplexing strategy.

What strategies should researchers employ when contradictory results are obtained with different LRRN3 antibodies?

Resolving contradictory results requires a systematic approach:

• Epitope mapping comparison:

  • Identify the specific regions targeted by each antibody

  • Consider whether different splice variants or post-translational modifications might affect epitope recognition

• Validation hierarchy assessment:

  • Evaluate the validation level of each antibody (supplier validation vs. peer-reviewed publications)

  • Prioritize results from antibodies with more extensive validation

• Independent verification methods:

  • Implement orthogonal techniques (e.g., mRNA expression, mass spectrometry)

  • Generate knockdown/knockout controls to test each antibody

  • Use tagged recombinant LRRN3 expression systems

• Methodological standardization:

  • Ensure identical experimental conditions when comparing antibodies

  • Document all protocol variations that might contribute to discrepancies

• Collaborative cross-validation:

  • Exchange samples with other labs studying LRRN3

  • Participate in antibody validation initiatives or consortia

For publication, transparently report all antibodies tested and potential explanations for discrepancies rather than selectively reporting confirmatory results.