LCR31 Antibody

What is LRRC31 and why is it a target for antibody development?

LRRC31 (Leucine-rich repeat-containing protein 31, also known as UNQ9367/PRO34156) is a human protein with a UniProt ID of Q6UY01. The protein contains leucine-rich repeat domains, which are structural motifs often involved in protein-protein interactions. Antibodies targeting LRRC31 are developed to enable detection, localization, and functional characterization of this protein in various experimental contexts. While the specific biological function of LRRC31 remains under investigation, the protein family to which it belongs is involved in diverse cellular processes including signal transduction, cell adhesion, and immune responses .

What applications are LRRC31 antibodies validated for?

Current commercially available LRRC31 antibodies, such as the FITC-conjugated polyclonal antibody, have been validated primarily for ELISA applications. The antibody is generated using recombinant human LRRC31 protein (amino acids 1-300) as an immunogen. It's important to note that many LRRC31 antibodies have not yet been extensively tested in other applications such as Western blotting, immunohistochemistry, or flow cytometry . Researchers should perform their own validation experiments when applying these antibodies to new methodological contexts.

How should LRRC31 antibodies be stored to maintain reactivity?

LRRC31 antibodies should be shipped at 4°C, and upon delivery, should be aliquoted to prevent repeated freeze-thaw cycles. Long-term storage should be at -20°C or -80°C. The antibody is typically provided in a liquid form containing preservatives (such as 0.03% Proclin 300) and stabilizers (such as 50% Glycerol in 0.01M PBS, pH 7.4) . Researchers should strictly follow the storage recommendations to maintain antibody performance, as improper storage can lead to degradation of the antibody and loss of specific binding capacity .

What controls should be included when using LRRC31 antibodies in experiments?

When using LRRC31 antibodies, researchers should include multiple controls to ensure experimental validity:

• Positive control: Cell lines or tissues known to express LRRC31

• Negative control: Cell lines with confirmed absence of LRRC31 expression or LRRC31 knockout models

• Isotype control: Rabbit IgG (for polyclonal LRRC31 antibodies) at the same concentration to evaluate non-specific binding

• Secondary antibody-only control: To assess background signal

• Blocking peptide control: Where the antibody is pre-incubated with excess immunizing peptide to confirm specificity

These controls help distinguish specific signals from background and non-specific binding, which is essential for accurate interpretation of results.

How can I address cross-reactivity concerns with LRRC31 antibodies?

Cross-reactivity is a significant concern with antibodies against leucine-rich repeat proteins due to structural similarities within this protein family. To address potential cross-reactivity of LRRC31 antibodies:

• Perform competitive binding assays with recombinant LRRC31 and structurally similar proteins

• Test the antibody on knockout or knockdown cell lines lacking LRRC31 expression

• Compare binding patterns across multiple LRRC31 antibodies targeting different epitopes

• Use orthogonal methods (like mass spectrometry) to confirm target identity

• Consider using transcriptomics to correlate antibody staining with mRNA expression patterns

For comprehensive validation, researchers should employ knockout cell lines specifically for LRRC31, as this approach provides the most definitive evidence for antibody specificity. Mass spectrometry analysis of immunoprecipitated proteins can further confirm target identity.

What are the considerations for using FITC-conjugated LRRC31 antibodies in multi-color flow cytometry experiments?

When incorporating FITC-conjugated LRRC31 antibodies into multi-color flow cytometry panels, researchers should consider:

• Spectral overlap: FITC (excitation ~490nm, emission ~525nm) shows significant overlap with other green fluorophores like PE and GFP. Proper compensation controls are essential.

• Panel design:

• Fixation effects: Aldehyde-based fixatives can increase autofluorescence in the FITC channel. Test fixation protocols to optimize signal-to-noise ratio.

• Alternative conjugates: If LRRC31 signal is weak or masked by autofluorescence, consider testing antibodies with brighter fluorophores (e.g., PE, APC) or those with different excitation/emission profiles .

How can I generate recombinant monoclonal antibodies against LRRC31 to improve reproducibility?

Generating recombinant monoclonal antibodies against LRRC31 follows a systematic approach:

• Sequence determination of existing hybridoma-derived antibodies:

  • If starting with an existing monoclonal antibody, sequence it using tandem mass spectrometry with W-ion isoleucine and leucine determination

  • Identify hypervariable regions (HVR/CDRs), framework regions, and constant regions

• Geneblock synthesis and vector preparation:

  • Design geneblocks encoding heavy chain (HC) and light chain (LC) sequences optimized for expression in human cells

  • Clone sequences into appropriate expression vectors with signal peptide sequences for secretion

  • Co-transfect vectors at an optimal ratio (typically 2:3 HC:LC) into suspension HEK293 cells

• Purification and validation:

  • Collect cell supernatant 5 days post-transfection

  • Purify antibody using Protein A or G Sepharose columns

  • Validate binding specificity through multiple applications

  • Compare performance against original antibody source

• Species switching and fragment generation:

  • For improved versatility, constant regions can be swapped to generate antibodies recognized by different species' secondary antibodies

  • Generate antibody fragments (scFv, Fab, scFvC) for specialized applications requiring smaller binding molecules

This approach yields renewable, sequence-defined antibodies with consistent performance across batches, addressing a major limitation of hybridoma-derived antibodies.

What epitope mapping strategies are recommended for LRRC31 antibodies?

Understanding the specific epitope recognized by LRRC31 antibodies is crucial for experimental design and interpretation. Recommended epitope mapping strategies include:

• Peptide array analysis:

  • Generate overlapping peptides spanning the LRRC31 sequence

  • Assess antibody binding to identify linear epitopes

  • Focus particularly on the 1-300AA region used as immunogen for many commercial antibodies

• Deletion/truncation mutants:

  • Express truncated versions of LRRC31 protein

  • Test antibody binding to narrow down the epitope region

  • This approach is particularly useful for conformational epitopes

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium uptake of LRRC31 alone versus antibody-bound

  • Reduced deuterium incorporation indicates protected regions (epitope)

  • Provides high-resolution mapping of conformational epitopes

• Cryo-electron microscopy:

  • For detailed structural characterization of antibody-antigen complexes

  • Reveals precise atomic interactions at the binding interface

Epitope information helps predict potential cross-reactivity issues, design blocking experiments, and determine if the antibody will recognize denatured protein in applications like Western blotting.

How should LRRC31 antibodies be validated for immunofluorescence applications?

While current commercial LRRC31 antibodies are primarily validated for ELISA, researchers extending their use to immunofluorescence should implement a comprehensive validation strategy:

• Expression system validation:

  • Test cells with confirmed LRRC31 expression (via RNA-seq or qPCR)

  • Include negative control cells (ideally LRRC31 knockout)

  • Compare staining patterns with subcellular localization predictions

• Titration optimization:

  • Test a concentration series (typically 0.1-10 μg/ml)

  • Evaluate signal-to-noise ratio at each concentration

  • Determine optimal antibody concentration that maximizes specific signal while minimizing background

• Specificity controls:

  • Pre-adsorption with immunizing peptide should abolish specific staining

  • Different fixation methods may affect epitope accessibility (try paraformaldehyde, methanol, and glutaraldehyde)

  • Test multiple LRRC31 antibodies targeting different epitopes if available

• Co-localization studies:

  • Perform dual staining with markers of predicted subcellular compartments

  • Quantify co-localization using appropriate statistical measures

For comprehensive validation, document all experimental parameters including fixation method, blocking reagents, antibody concentration, incubation time/temperature, and image acquisition settings .

What are the optimal conditions for using LRRC31 antibodies in ELISA?

ELISA is the primary validated application for many commercial LRRC31 antibodies. To optimize ELISA performance:

• Coating conditions:

  • For direct ELISA: Coat with recombinant LRRC31 at 1-5 μg/ml in carbonate buffer (pH 9.6)

  • For sandwich ELISA: Use capture antibody against different LRRC31 epitope than detection antibody

• Blocking optimization:

  • Test different blocking agents (BSA, casein, commercially available blockers)

  • Typically use 1-5% blocker in PBS or TBS

• Antibody titration:

  • Generate a titration curve with 2-fold serial dilutions

  • Plot signal-to-noise ratio against antibody concentration

  • Select concentration at upper end of linear range

• Sample preparation:

  • For cell lysates: Use non-denaturing lysis buffers to preserve native epitopes

  • For serum/plasma: Consider pre-clearing with Protein A/G to reduce background

• Detection system:

  • For FITC-conjugated antibodies: Use anti-FITC detection systems or direct fluorescence readout

  • For unconjugated antibodies: Use species-appropriate enzyme-conjugated secondary antibodies

• Quantification:

  • Always include a standard curve with recombinant LRRC31 protein

  • Report results as absolute concentrations rather than arbitrary units when possible

How do batch-to-batch variations affect LRRC31 antibody experiments?

Batch-to-batch variations are a critical consideration in antibody-based research and can significantly impact experimental reproducibility:

• Sources of variation:

  • Production process differences (especially for polyclonal antibodies)

  • Changes in immunization protocols or antigen preparation

  • Purification method inconsistencies

  • Storage condition variations

• Performance metrics to monitor:

• Mitigation strategies:

  • Purchase larger lots when possible to minimize batch changes

  • Maintain internal reference standards for new batch qualification

  • Document lot numbers used in all experiments

  • Consider switching to recombinant antibodies for improved consistency

• Recombinant alternatives:

  • If batch variations cause significant research delays, consider developing recombinant LRRC31 antibodies

  • While initially more time-consuming, recombinant antibodies provide superior long-term reproducibility through sequence-defined reagents

How can I ensure reproducibility when using LRRC31 antibodies across different experimental systems?

Ensuring reproducibility with LRRC31 antibodies requires systematic documentation and standardization:

• Comprehensive documentation:

  • Record complete antibody information: supplier, catalog number, lot number, clone (if monoclonal), host species, and antigen sequence

  • Document all experimental conditions in sufficient detail for reproduction

  • Maintain validation data for each application and experimental system

• Standardized protocols:

  • Develop detailed standard operating procedures (SOPs) for each application

  • Include all buffer compositions, incubation times/temperatures, and equipment settings

  • Implement consistent sample preparation methods

• Cross-platform validation:

  • When transitioning between systems (e.g., different cell types or tissue sources), revalidate antibody performance

  • Compare staining patterns and signal intensities across systems

  • Adjust protocols as needed while maintaining core validation controls

• Data sharing practices:

  • Submit antibody validation data to repositories like Antibodypedia or CiteAb

  • Include complete methodology in publications rather than abbreviated methods

  • Consider open notebook science approaches for sharing detailed protocols

Reproducibility challenges often arise from insufficient methodological detail rather than actual irreproducibility. Thorough documentation of antibody characteristics and experimental conditions is essential for reproducible research.

What strategies can address non-specific binding of LRRC31 antibodies?

Non-specific binding can confound experimental results when using LRRC31 antibodies. Effective troubleshooting strategies include:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, normal serum from secondary antibody species, commercial blockers)

  • Evaluate longer blocking times or higher blocker concentrations

  • Consider adding protein carriers (0.1-0.5% BSA) to antibody dilution buffers

• Buffer modifications:

  • Add non-ionic detergents (0.05-0.3% Triton X-100 or Tween-20) to reduce hydrophobic interactions

  • Include salt (150-500 mM NaCl) to disrupt low-affinity ionic interactions

  • Test different pH conditions to optimize specific binding

• Pre-adsorption strategies:

  • Pre-incubate antibody with tissues or cell lysates lacking the target to remove cross-reactive antibodies

  • For tissue sections, consider pre-blocking with endogenous biotin/avidin if using biotin-based detection systems

• Signal amplification alternatives:

  • If using signal amplification methods (like tyramide), reduce primary antibody concentration

  • Consider direct detection methods when non-specific binding persists despite optimization

• Isotype-matched control experiments:

  • Use proper concentration-matched isotype controls to identify non-specific binding

  • Subtract isotype control signal during analysis when appropriate

Systematic optimization of these parameters should be documented to establish robust protocols that minimize non-specific binding while maintaining sensitive detection of LRRC31.

How does antibody validation data contribute to research integrity when working with LRRC31?

Proper antibody validation is fundamental to research integrity and reproducibility:

• Validation as quality assurance:

  • Validation data provides evidence that experimental observations are attributable to the intended target

  • Without validation, results may reflect artifactual or non-specific effects rather than LRRC31 biology

  • Well-validated antibodies allow meaningful comparison of results across studies and laboratories

• Validation hierarchy:

• Reporting standards:

  • Comprehensive validation data should be included in publications or supplementary materials

  • Negative results from validation experiments should be reported to prevent others from repeating problematic approaches

  • Explicit acknowledgment of validation limitations helps appropriate interpretation of results

• Continuous validation:

  • Validation should be viewed as an ongoing process rather than a one-time effort

  • Each new experimental context requires at least partial revalidation

  • Unexpected results should trigger additional validation experiments rather than immediate biological interpretation

The scientific community increasingly recognizes that poor antibody validation undermines research integrity and contributes significantly to the reproducibility crisis. Thorough validation of LRRC31 antibodies is therefore an ethical imperative, not merely a technical consideration.

What are the future directions for improving LRRC31 antibody research?

Advancing LRRC31 antibody research will require coordinated efforts across several domains:

• Development of recombinant antibodies:

  • Sequence-defined recombinant antibodies would provide superior reproducibility

  • Generation of diverse antibody formats (full-length, Fab, scFv) would expand application range

  • Species-agnostic variants would facilitate cross-species comparisons

• Comprehensive epitope mapping:

  • Detailed characterization of binding sites would aid interpretation of functional studies

  • Multiple antibodies targeting distinct epitopes would enable confirmation of results

  • Structure-function analyses could connect antibody binding to biological effects

• Integrated validation approaches:

  • Combining orthogonal methods (proteomics, transcriptomics) with antibody detection

  • Development of LRRC31 knockout cell lines as definitive negative controls

  • Community-based validation efforts to distribute workload and standardize practices

• Enhanced data sharing:

  • Centralized repositories for LRRC31 antibody validation data

  • Standardized reporting formats to facilitate comparison across studies

  • Implementation of unique antibody identifiers to track reagent provenance

• Application expansion:

  • Validation beyond ELISA to include Western blotting, immunohistochemistry, and flow cytometry

  • Development of application-specific protocols optimized for LRRC31 detection

  • Cross-platform validation to ensure consistent performance across methods