lrrc3 Antibody

What is LRRC3 and why is it significant in research contexts?

LRRC3 (Leucine-rich repeat-containing protein 3) is a 25 kDa member of the Leucine-rich repeat protein superfamily. The human LRRC3 protein contains 225 amino acids with three distinct LRRs between amino acids 63-86, 87-110, and 112-135. It shows widespread tissue expression but appears to be particularly prominent in neuronal tissues, especially in the brain medulla region.

While the complete functional characterization of LRRC3 remains ongoing, research indicates its potential significance in:

• Neuronal development and function

• Possible roles in immune regulation

• Expression pattern alterations in pathological states

The human LRRC3 protein shares 83% amino acid identity with mouse LRRC3 and 81% with canine LRRC3 (over amino acids 33-204), suggesting evolutionary conservation of this protein .

What types of LRRC3 antibodies are currently available for research applications?

Several types of LRRC3 antibodies are available for research, including:

Most commercially available antibodies target specific epitopes within the LRRC3 protein, with some targeting recombinant protein fragments corresponding to amino acids 59-236 or other regions containing the leucine-rich repeats. Several antibodies have been validated through multiple techniques including Western blot, immunohistochemistry, and protein arrays containing the target protein plus hundreds of non-specific proteins to ensure specificity .

How should researchers determine optimal dilutions when using LRRC3 antibodies for different experimental applications?

Determining optimal antibody dilution requires empirical testing for each specific application, but general guidelines based on validated protocols include:

For Western Blot:

• Starting dilution range: 0.04-0.4 μg/mL (for purified antibodies)

• Alternatively: 1:1000-1:5000 dilution from commercial stock concentrations

• Protocol adjustment: Increase antibody concentration if signal is weak; decrease if background is high

For Immunohistochemistry:

• Paraffin sections: 1:100-1:500 dilution (or 10-15 μg/mL for purified antibodies)

• Optimal antigen retrieval: Heat-induced epitope retrieval using basic buffer (pH 9.0)

• Detection system: Anti-species HRP-DAB systems show good results with LRRC3 antibodies

For Immunofluorescence:

• Starting dilution: 1:100-1:200

• Fixation method: 4% paraformaldehyde shows best results with LRRC3 antibodies

Always perform a dilution series during optimization and include both positive controls (tissues known to express LRRC3, such as brain medulla) and negative controls (IgG matched to the host species of the primary antibody) .

What are the recommended protocols for using LRRC3 antibodies in Western blot analysis?

Optimized Western Blot Protocol for LRRC3 Detection:

• Sample Preparation:

  • For brain tissue: Prepare lysates in RIPA buffer with protease inhibitors

  • Expected molecular weight: ~115 kDa (observed in human brain hypothalamus tissue)

• Electrophoresis Conditions:

  • Use reducing conditions (include β-mercaptoethanol in sample buffer)

  • 8-10% SDS-PAGE gels recommended due to protein size

• Transfer Settings:

  • PVDF membrane shows better results than nitrocellulose for LRRC3

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and Antibody Incubation:

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

  • Primary antibody: Dilute to 1 μg/mL in blocking buffer, incubate overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-species IgG at 1:2000-1:5000 dilution

• Detection:

  • Enhanced chemiluminescence (ECL) systems work well

  • Expected band: Clear band at approximately 115 kDa

This protocol is based on published methodologies showing successful detection of LRRC3 in human brain hypothalamus tissue using Mouse Anti-Human LRRC3 Monoclonal Antibody (MAB69191) .

What are the validated procedures for LRRC3 immunohistochemistry in neural tissues?

Validated IHC Protocol for LRRC3 Detection in Brain Tissues:

• Tissue Preparation and Sectioning:

  • 10% formalin-fixed, paraffin-embedded sections at 4-6 μm thickness

  • Mount on positively charged slides

• Deparaffinization and Antigen Retrieval:

  • Standard deparaffinization through xylene and graded alcohols

  • Critical step: Heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (pH 9.0)

  • Heating method: Pressure cooker for 20 minutes or water bath at 95-98°C for 20-30 minutes

• Blocking and Antibody Application:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Protein block: 5% normal serum from the same species as secondary antibody

  • Primary antibody: Apply LRRC3 antibody at 10-15 μg/mL (or 1:100-1:200 dilution)

  • Incubation time: Overnight at 4°C or 1 hour at room temperature

• Detection System:

  • HRP-polymer detection systems show superior results compared to avidin-biotin methods

  • Chromogen: DAB (3,3'-diaminobenzidine) with hematoxylin counterstain

• Expected Results:

  • Positive staining: Neuronal cell bodies in human brain medulla

  • Subcellular localization: Primarily cytoplasmic

This protocol has been validated with both polyclonal (AF5039) and monoclonal (MAB5039) antibodies on human brain tissue sections, specifically showing localization to neuronal cytoplasm in the medulla region .

How do LRRC3 antibodies contribute to understanding TGF-β signaling in cancer immunotherapy?

Recent research has revealed connections between the LRRC family and TGF-β signaling, particularly through the paralog LRRC33. While LRRC33 (not LRRC3) has been shown to be specifically associated with TGF-β1 and is required for surface display and activation of TGF-β1 on tumor-infiltrating myeloid cells, this research provides important context for studying the broader LRRC family .

Key findings from cancer immunotherapy research using LRRC family antibodies:

• LRRC33-TGF-β1 axis in tumor microenvironment:

  • Loss of LRRC33-dependent TGF-β1 activation slowed tumor growth and metastasis

  • Enhanced both innate and adaptive anti-tumor immunity in multiple mouse tumor models

  • Created a more immunogenic microenvironment with:

    • Decreased myeloid-derived suppressor cells

    • More active CD8+ T and NK cells

    • Skewing toward tumor-suppressive M1 macrophages

• Synergistic effects with checkpoint inhibitors:

  • LRRC33 loss and PD-1 blockade showed synergistic effects in controlling B16.F10 tumor growth

  • This suggests potential for dual blockade approaches in cancer immunotherapy

• Methodological approach for similar LRRC3 studies:

  • Generate knockout models to assess functional significance

  • Use flow cytometry to evaluate immune cell populations

  • Employ antibodies for both in vivo modulation and ex vivo analysis

While these findings focus on LRRC33, they provide methodological frameworks and biological insights that could be applied to investigating potential roles of LRRC3 in similar contexts .

What experimental approaches can be used to investigate LRRC3 expression patterns across normal versus pathological tissues?

Multi-modal Approach to LRRC3 Expression Analysis:

• Immunohistochemistry on Tissue Microarrays:

  • Method: Use validated LRRC3 antibodies (e.g., HPA017975) on tissue microarrays containing:

    • 44 normal human tissues from different organs

    • 20 common cancer types

  • Analysis: Semi-quantitative scoring of staining intensity and percentage of positive cells

  • Advantage: Provides spatial information and cellular localization

• Western Blot Quantification:

  • Method: Prepare tissue lysates from matched normal/pathological samples

  • Analysis: Densitometric quantification of 115 kDa LRRC3 band normalized to loading controls

  • Controls: Include recombinant LRRC3 protein as positive control

• Transcriptomic Correlation:

  • Method: Correlate protein expression data with RNA-seq or microarray data

  • Analysis: Calculate protein-mRNA correlation coefficients to identify potential post-transcriptional regulation

• Single-cell Analysis:

  • Method: Single-cell immunofluorescence or mass cytometry with LRRC3 antibodies

  • Analysis: Identify cell-type specific expression patterns and heterogeneity within tissues

This multi-modal approach has been implemented in studies of brain tissue, where LRRC3 shows specific localization to neuronal cell bodies, particularly in the medulla region .

How can researchers effectively use LRRC3 antibodies in co-localization studies with other neuronal markers?

Protocol for LRRC3 Co-localization Studies in Neuronal Tissues:

• Sample Preparation:

  • Optimized fixation: 4% paraformaldehyde for 15-20 minutes

  • Permeabilization: 0.2% Triton X-100 for 10 minutes

• Multiple Labeling Strategy:

  • Primary antibody combinations:

    • LRRC3 antibody (rabbit or mouse based on compatibility)

    • Neuronal markers (NeuN, MAP2, β-III-tubulin)

    • Glial markers (GFAP, Iba1, Olig2) as controls

  • Use antibodies raised in different species to avoid cross-reactivity

• Sequential Staining Approach:

  • For same-species antibodies: Use sequential staining with intermediate blocking

  • For different-species antibodies: Apply simultaneously

• Controls for Co-localization Studies:

  • Single-stained controls for spectral bleed-through assessment

  • Isotype controls for each primary antibody

  • Absorption controls using recombinant LRRC3 protein

• Imaging and Analysis:

  • Capture Z-stacks using confocal microscopy

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Three-dimensional reconstruction for spatial relationship analysis

This approach has been used to determine that LRRC3 is predominantly expressed in neuronal cell bodies but not in glial populations, providing important spatial context for functional studies .

How can researchers address non-specific binding and background issues when using LRRC3 antibodies?

Comprehensive Troubleshooting Strategy for LRRC3 Antibody Background Issues:

• Antibody Validation Before Experiments:

  • Verify antibody specificity using Western blot on tissues known to express LRRC3

  • Pre-absorption test: Pre-incubate antibody with recombinant LRRC3 protein

  • Test multiple antibodies targeting different epitopes if possible

• For Western Blot Background Issues:

  • Increase washing duration and number of washes (5x 10 minutes with TBST)

  • Optimize primary antibody concentration using titration (0.04-0.4 μg/mL range)

  • Increase blocking agent concentration to 5-10%

  • Use specialized blocking agents (e.g., SuperBlock™ or commercial protein-free blockers)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

• For Immunohistochemistry Background:

  • Test multiple antigen retrieval methods (heat vs. enzymatic)

  • Include 10-15 minute treatment with 0.3% H₂O₂ in methanol to block endogenous peroxidase

  • Add avidin/biotin blocking step if using biotin-based detection systems

  • Use species-specific blocking serums matched to secondary antibody

• For Immunofluorescence Optimization:

  • Include Sudan Black B treatment (0.1-0.3%) to reduce autofluorescence

  • Optimize fixation time to minimize epitope masking

  • Use directly conjugated primary antibodies to eliminate secondary antibody issues

• Additional Controls:

  • Include isotype controls at the same concentration as primary antibody

  • Use tissues from LRRC3 knockout models if available

  • Perform secondary-only controls to assess non-specific binding

These protocols were derived from successful applications of LRRC3 antibodies in neuronal tissues and can significantly improve signal-to-noise ratio in challenging experimental contexts .

What are the recommended storage and handling procedures to maximize LRRC3 antibody stability and performance?

Optimal Storage and Handling Protocol for LRRC3 Antibodies:

• Long-term Storage Conditions:

  • Temperature: -20°C to -70°C for unopened/stock antibodies

  • Storage format: Avoid repeated freeze-thaw cycles by making small aliquots

  • For lyophilized antibodies: Reconstitute in 100 μL sterile distilled water with 50% glycerol

• Short-term Storage (1 month):

  • Temperature: 2-8°C under sterile conditions after reconstitution

  • Avoid exposure to light for fluorochrome-conjugated antibodies

  • Add preservatives if diluting for repeated use (0.02% sodium azide for non-enzymatic applications)

• Working Solution Preparation:

  • Always centrifuge antibody vial briefly before opening (30 seconds at 10,000g)

  • Use sterile techniques when handling antibody solutions

  • Prepare working dilutions immediately before use when possible

  • If working dilutions must be stored, keep at 4°C for maximum of 1 week

• Stability Timeframes:

  • Unopened/stock: 12 months from date of receipt at -20°C to -70°C

  • After reconstitution:

    • 1 month at 2-8°C

    • 6 months at -20°C to -70°C under sterile conditions

• Transportation Guidelines:

  • Transport on ice packs for short distances

  • Use dry ice for overnight or longer shipments

  • Avoid exposure to direct sunlight or extreme temperatures

Following these storage and handling procedures significantly extends the shelf life and performance of LRRC3 antibodies. Most commercially available LRRC3 antibodies maintain their activity for at least 12 months when stored properly at -20°C to -70°C .

How are LRRC3 antibodies being used in neurodegenerative disease research?

LRRC3 antibodies are emerging as valuable tools in neurodegenerative disease research, particularly given the protein's expression in neuronal tissues. Current applications include:

• Expression Profiling in Pathological States:

  • LRRC3 antibodies enable comparison of expression patterns between normal and diseased brain tissues

  • IHC studies have demonstrated LRRC3 localization to neuronal cell bodies in the medulla region

  • Changes in expression patterns may serve as biomarkers for specific neurodegenerative conditions

• Potential Role in TGF-β Signaling Pathways:

  • Based on research on the related family member LRRC33, researchers are investigating whether LRRC3 plays a similar role in TGF-β regulation

  • Patients deficient in LRRC33 show severe infantile-onset neurodegeneration, suggesting potential similar roles for other LRRC family members

• Methodological Approaches in Current Research:

  • Immunoprecipitation to identify protein-protein interactions

  • Comparative expression analysis across brain regions in neurodegenerative disease models

  • Co-localization with known disease markers (tau, amyloid, α-synuclein)

• Research Directions for LRRC3 in Neurodegeneration:

  • Investigation of LRRC3 expression changes in Alzheimer's and Parkinson's disease tissues

  • Development of proximity ligation assays to detect LRRC3 interactions with disease-relevant proteins

  • Creation of conditional knockout models to assess functional contributions to disease progression

While specific findings linking LRRC3 to neurodegenerative diseases are still emerging, researchers are actively using LRRC3 antibodies to explore potential roles in neuronal function and pathology .

What considerations should researchers make when designing comparative studies of LRRC family members (LRRC3, LRRC4, LRRC33)?

Experimental Design Principles for Comparative LRRC Family Studies:

• Antibody Specificity Assessment:

  • Critical issue: LRRC family members share sequence homology

  • Solution: Validate antibody specificity using:

    • Western blot analysis of recombinant proteins for each LRRC family member

    • Knockout/knockdown controls for each family member

    • Epitope mapping to identify unique targeting regions

• Cross-Species Comparison Considerations:

  • Sequence conservation: Human LRRC3 shares 83% amino acid identity with mouse LRRC3

  • Experimental approach: Use multi-species tissue panels to identify conserved versus species-specific patterns

  • Analysis method: Phylogenetic analysis of expression patterns across species

• Functional Differentiation Strategy:

  • LRRC4B/NGL-3 (not LRRC3) contains nine LRRs, C2-type Ig-like domains, and recognizes receptor tyrosine phosphatases

  • LRRC33 specifically associates with TGF-β1 (not TGF-β2/3)

  • Comparative approach: Use co-immunoprecipitation studies to identify unique binding partners

  • Analytical technique: Proximity ligation assays to visualize protein-protein interactions in situ

• Expression Pattern Analysis:

  • Method: Parallel IHC/IF staining with specific antibodies for each family member

  • Analysis: Co-expression analysis at tissue, cellular, and subcellular levels

  • Data integration: Correlation of protein expression with transcriptomic data

• Structural-Functional Correlation:

  • Challenge: Distinguishing unique versus redundant functions

  • Approach: Domain-specific antibodies targeting unique structural features

  • Application: Functional blocking studies to identify domain-specific activities

These methodological considerations are essential for accurate differentiation between LRRC family members, particularly given their structural similarities but potentially distinct functions, as evidenced by the specific TGF-β1 interaction profile of LRRC33 versus other family members .

What recent technological advances are improving LRRC3 antibody development and application in research?

Technological Innovations Enhancing LRRC3 Antibody Research:

• Advanced Antibody Generation Methods:

  • Recombinant antibody technology: Production of highly specific monoclonal antibodies against defined LRRC3 epitopes

  • Phage display selection: Isolation of antibodies with superior affinity and specificity

  • Rational epitope design: Targeting non-conserved regions to minimize cross-reactivity with other LRRC family members

• Validation Technologies:

  • Enhanced validation using knockout controls: Confirming antibody specificity through genetic models

  • Protein arrays: Testing antibodies against panels of 384+ proteins to ensure specificity

  • Tandem mass spectrometry validation: Confirming antibody targets through orthogonal protein identification methods

• Imaging and Detection Innovations:

  • Super-resolution microscopy: Enabling subcellular localization studies with 20-50nm resolution

  • Multiplexed immunofluorescence: Simultaneous detection of LRRC3 with multiple markers

  • Spatial transcriptomics integration: Correlating protein expression with gene expression at the tissue level

• Functional Application Advances:

  • Proximity-dependent biotinylation (BioID): Identifying proximal protein interactors

  • Intrabodies: Expressing antibody fragments intracellularly to visualize or modulate LRRC3 function

  • CRISPR epitope tagging: Endogenous tagging for antibody-independent detection and purification

• Bioinformatic Integration:

  • Antibody epitope mapping databases: Predicting cross-reactivity through sequence analysis

  • Structure-based epitope prediction: Improving antibody design through 3D protein modeling

  • Machine learning approaches: Predicting optimal applications based on antibody characteristics

These technological advances are significantly improving the quality, specificity, and research applications of LRRC3 antibodies, enabling more sophisticated studies of this protein's function in various physiological and pathological contexts .

How should researchers approach cross-species validation when using LRRC3 antibodies in animal models?

Comprehensive Cross-Species Validation Protocol:

• Sequence Homology Analysis:

  • Perform sequence alignment between human LRRC3 and target species (mouse, rat, non-human primates)

  • Focus on epitope regions recognized by the antibody

  • Critical data: Human LRRC3 shares 83% amino acid identity with mouse LRRC3 and 81% with canine LRRC3 (over aa 33-204)

• Stepwise Validation Approach:

  • Phase 1: Western blot analysis

    • Run purified recombinant LRRC3 from multiple species

    • Test tissue lysates from equivalent organs across species

    • Verify molecular weight differences (human: ~25 kDa predicted)

  • Phase 2: Immunohistochemistry validation

    • Perform parallel IHC on fixed tissues from multiple species

    • Compare staining patterns, focusing on neuronal tissues

    • Use identical protocols to enable direct comparison

  • Phase 3: Functional validation

    • Perform immunoprecipitation studies to confirm target binding

    • Use blocking peptides specific to each species

    • Test antibody on knockout tissues when available

• Non-human Primate Specific Considerations:

  • Rhesus macaque (Macaca mulatta) LRRC3 has been characterized at the genomic level

  • Available database information includes multiple transcript variants (XM_015132715.2, NM_001194240.1)

  • Test antibodies on rhesus tissues before conducting larger primate studies

This methodological approach enables researchers to confidently use LRRC3 antibodies across species, understanding the limitations and strengths of cross-species reactivity .

What strategies can optimize detection of low-abundance LRRC3 in challenging tissue types?

Advanced Detection Strategies for Low-Abundance LRRC3:

• Sample Enrichment Techniques:

  • Subcellular fractionation to concentrate compartments where LRRC3 is expressed

  • Immunoprecipitation before Western blot for signal amplification

  • Laser capture microdissection to isolate specific LRRC3-expressing cells

• Signal Amplification Methods for IHC/IF:

  • Tyramide signal amplification (TSA): Offers 10-100× signal enhancement

  • Polymer-based detection systems rather than traditional avidin-biotin methods

  • Multiple antibody layer approaches (e.g., bridging antibodies between primary and detection system)

• Protocol Optimization for Western Blot:

  • Extended primary antibody incubation (overnight at 4°C)

  • Higher primary antibody concentration (up to 1-2 μg/mL for low abundance targets)

  • Enhanced chemiluminescence substrates with extended signal duration

  • PVDF membranes (rather than nitrocellulose) for higher protein binding capacity

• Advanced Microscopy Techniques:

  • Spectral imaging to differentiate specific signal from autofluorescence

  • Long exposure capture with cooled CCD cameras

  • Deconvolution algorithms to improve signal-to-noise ratio

  • Combining results from multiple antibodies targeting different LRRC3 epitopes

• Validation of Low-Abundance Signals:

  • Parallel detection with multiple antibodies against different epitopes

  • Correlation with mRNA expression data (ISH or RNA-seq)

  • Depletion/enrichment controls to confirm specificity

These techniques have proven effective for detecting low-abundance proteins in neuronal tissues and can be applied specifically to LRRC3 detection in challenging samples .