LENG9 Antibody

What is LENG9 and why is it a target for antibody development?

LENG9 (leukocyte receptor cluster member 9) is a protein of significant research interest in human immunology and cell biology. In humans, the canonical protein consists of 501 amino acid residues with a molecular mass of 53.2 kDa. It belongs to the leukocyte receptor cluster (LRC) family and is widely expressed across multiple tissues, with notable expression in the bronchus, vagina, and appendix tissues . The protein's extensive tissue distribution suggests potential functional importance in various biological processes, making it a valuable target for antibody development to study its expression patterns, interactions, and possible roles in normal and pathological conditions.

What applications are LENG9 antibodies most commonly used for?

LENG9 antibodies are employed across multiple immunodetection applications in research settings. The most widely used applications include:

• Immunohistochemistry (IHC): For detecting LENG9 expression in tissue sections

• Western Blot (WB): For protein identification and semi-quantitative analysis

• Immunofluorescence (IF): For subcellular localization studies

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection

The selection of application depends on the specific research question, with many antibodies being validated for multiple techniques. Most commercial LENG9 antibodies are specifically tested for human reactivity, though orthologs have been identified in mouse, rat, bovine, frog, zebrafish, and chimpanzee models .

What is the recommended methodology for validating a new LENG9 antibody?

When validating a new LENG9 antibody for research use, a comprehensive approach following established validation procedures is essential. A recommended methodological workflow includes:

• Cell line selection: Use proteomic databases like PaxDB to identify cell lines with relatively high LENG9 expression that are amenable to genetic modification .

• CRISPR/Cas9 knockout: Generate a LENG9 knockout in the selected cell line to create a definitive negative control .

• Initial screening: Test the antibody by immunoblot comparing parental and knockout cell lines to verify specificity .

• Expression analysis: Perform quantitative immunoblots on a panel of cell lines to identify those with the highest LENG9 expression levels for subsequent validation experiments .

• Advanced validation: Use the validated cell lines to screen for antibody specificity in:

  • Immunoprecipitation assays

  • Immunofluorescence studies

  • Additional tissue-specific applications as needed

This methodical approach ensures antibody specificity and reduces the risk of misinterpreting results due to non-specific binding.

How should researchers determine the optimal working dilution for LENG9 antibodies in different applications?

Determining the optimal working dilution for LENG9 antibodies requires a systematic titration approach specific to each application. While manufacturer recommendations provide starting points (e.g., 1:50-1:200 for immunohistochemistry and 0.25-2 μg/mL for immunofluorescence) , researchers should:

• Perform dilution series: Test a range of dilutions around the manufacturer's recommendation (e.g., 1:25, 1:50, 1:100, 1:200, 1:400 for IHC).

• Include controls: Always run positive controls (tissues known to express LENG9) and negative controls (LENG9 knockout tissues/cells and secondary-only controls).

• Evaluate signal-to-noise ratio: The optimal dilution provides the strongest specific signal with minimal background.

• Consider blocking optimization: If background remains problematic, test different blocking reagents (BSA, normal serum, commercial blockers) while maintaining the same antibody dilution.

• Document conditions: Record all experimental parameters, including sample preparation methods, incubation times/temperatures, and detection systems for reproducibility.

This methodical approach ensures reliable and specific LENG9 detection while conserving valuable antibody resources.

What are the critical factors that affect LENG9 antibody performance in immunohistochemistry?

Several critical factors can significantly impact LENG9 antibody performance in immunohistochemistry:

• Fixation method and duration: Overfixation can mask epitopes while underfixation can compromise tissue morphology. For LENG9 detection, standard 10% neutral buffered formalin fixation for 24-48 hours is generally appropriate, though optimization may be required.

• Antigen retrieval technique: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine which best exposes LENG9 epitopes without causing tissue degradation.

• Tissue section thickness: Optimal thickness is typically 4-5 μm; thicker sections may increase background while thinner sections may have insufficient antigen.

• Blocking protocol: Thorough blocking of endogenous peroxidase activity and non-specific binding sites is essential for clean results.

• Incubation conditions: Primary antibody incubation at 4°C overnight often produces better results than shorter incubations at room temperature.

• Detection system sensitivity: For low-abundance targets like LENG9, amplification systems such as polymer-based detection may yield better results than standard ABC methods.

• Counterstaining intensity: Overly intense hematoxylin counterstaining can obscure positive DAB staining, particularly for nuclear or low-expression targets.

Systematic optimization of these factors is necessary for reproducible LENG9 detection in tissue samples.

How can researchers effectively troubleshoot non-specific binding when using LENG9 antibodies?

When encountering non-specific binding with LENG9 antibodies, a structured troubleshooting approach should be implemented:

• Verify antibody specificity: Compare results with LENG9 knockout controls to confirm the observed pattern is truly non-specific binding rather than unexpected LENG9 expression .

• Optimize blocking: Increase blocking duration (60-90 minutes) and test alternative blocking agents (5% BSA, 5-10% normal serum matched to secondary antibody species, commercial blockers).

• Adjust antibody concentration: Dilute the primary antibody further if background is high while maintaining adequate incubation time.

• Modify washing steps: Increase the number and duration of washes (e.g., 5 washes × 5 minutes each) with gentle agitation in PBS-T (0.1% Tween-20).

• Pre-adsorb antibody: For polyclonal antibodies, pre-adsorption against tissue powder from the same species can reduce cross-reactivity.

• Test alternative buffers: Try different diluents for the primary antibody (PBS with 1% BSA, TBS with 0.1% Tween-20).

• Consider sample preparation: Some fixatives can increase background; test fresh samples or alternative fixation methods if possible.

• Evaluate secondary antibody: Test different lots or sources of secondary antibody, and ensure it's appropriately matched to the primary.

Systematic documentation of these optimization steps helps establish reliable protocols for future experiments.

What approaches can be used to study LENG9 protein interactions in cellular contexts?

Investigating LENG9 protein interactions requires sophisticated molecular techniques:

• Co-immunoprecipitation (Co-IP):

  • Use validated LENG9 antibodies to pull down LENG9 and associated proteins

  • Analyze precipitated complexes by mass spectrometry to identify interaction partners

  • Confirm interactions using reverse Co-IP with antibodies against identified partners

• Proximity Ligation Assay (PLA):

  • Enables detection of protein-protein interactions in situ with high sensitivity

  • Requires antibodies against LENG9 and suspected interaction partners from different species

  • Produces fluorescent signals only when proteins are within 40 nm of each other

• FRET/BRET Analysis:

  • For studying dynamic interactions in living cells

  • Requires fusion of fluorescent/bioluminescent proteins to LENG9 and potential partners

  • Measures energy transfer between proteins in close proximity

• Crosslinking Mass Spectrometry:

  • Chemical crosslinking stabilizes transient interactions

  • Crosslinked complexes containing LENG9 are purified and analyzed by mass spectrometry

  • Provides information about interaction interfaces

• Yeast Two-Hybrid Screening:

  • For systematic screening of potential LENG9 interaction partners

  • Can be followed by targeted validation using above methods in mammalian cells

Each method has strengths and limitations, and combining multiple approaches provides the most robust characterization of LENG9 interactomes.

How can researchers integrate LENG9 antibody data with genomic and transcriptomic findings?

Integrating LENG9 antibody-based protein data with genomic and transcriptomic findings requires a multi-omics approach:

• Correlation analysis:

  • Quantify LENG9 protein levels using validated antibodies across multiple samples

  • Measure LENG9 mRNA expression in the same samples

  • Calculate correlation coefficients to identify potential post-transcriptional regulation

• Cell type-specific expression:

  • Use LENG9 antibodies for immunohistochemistry or flow cytometry to identify cell populations expressing the protein

  • Compare with single-cell RNA-seq data to identify discrepancies suggesting post-transcriptional regulation

  • Create cell type-specific expression maps integrating both data types

• Response to genetic perturbations:

  • Analyze LENG9 protein expression changes following CRISPR-based modification of suspected regulatory elements

  • Compare with transcriptional responses to identify translation-specific effects

• Pathway integration:

  • Combine LENG9 antibody-based interaction data with pathway information from transcriptomics

  • Use network analysis tools to identify functional modules containing LENG9

  • Validate key nodes through targeted protein and transcript measurements

• Disease-associated variants:

  • Assess how genomic variants in LENG9 affect protein expression, localization, or interactions

  • Use LENG9 antibodies to characterize protein-level consequences of transcriptionally-detected alterations

This integrated approach provides a more complete understanding of LENG9 biology than any single data type alone.

What are the considerations for using LENG9 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence involving LENG9 antibodies requires careful planning and optimization:

• Antibody compatibility assessment:

  • Test for cross-reactivity between all antibodies in the multiplex panel

  • Ensure all primary antibodies are raised in different species or are of different isotypes

  • Verify that detection systems (secondary antibodies or direct conjugates) don't cross-react

• Spectral overlap management:

  • Select fluorophores with minimal spectral overlap

  • Consider brightness differences between targets (assign brighter fluorophores to less abundant targets like LENG9)

  • Include single-color controls for spectral unmixing during analysis

• Sequential staining protocol development:

  • Determine optimal order of antibody application (generally from weakest to strongest signal)

  • Test whether heat-mediated antibody stripping between rounds affects LENG9 epitope integrity

  • Validate complete stripping by re-probing with secondary antibodies alone

• Signal amplification considerations:

  • For low-abundance LENG9 detection, evaluate tyramide signal amplification (TSA) or other amplification methods

  • Ensure amplification doesn't increase background or cause signal bleeding into other channels

• Autofluorescence management:

  • Implement appropriate autofluorescence quenching protocols

  • Consider spectral unmixing algorithms during analysis to separate true signal from autofluorescence

• Validation controls:

  • Compare multiplex results with single-plex staining to ensure consistent LENG9 detection

  • Include biological controls (LENG9 knockout tissues) to confirm specificity in the multiplex context

These considerations enable reliable detection of LENG9 alongside other markers of interest in complex tissue samples.

How can researchers accurately quantify LENG9 expression levels in different tissues and cell types?

Accurate quantification of LENG9 expression across tissues and cell types requires a multi-modal approach:

• Western blot quantification:

  • Use validated LENG9 antibodies with recombinant LENG9 protein standards for absolute quantification

  • Normalize to total protein (measured by stain-free technology or housekeeping proteins)

  • Create standard curves spanning the expected range of LENG9 expression

  • Include the following quantification controls in each experiment:

• Immunohistochemistry quantification:

  • Use digital image analysis with validated algorithms for LENG9 detection

  • Implement H-score or other semi-quantitative scoring systems

  • Calibrate using cell lines with known LENG9 expression levels embedded in control blocks

• Flow cytometry:

  • Use directly conjugated LENG9 antibodies or validated secondary detection systems

  • Include quantification beads to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • Use median fluorescence intensity (MFI) for population comparisons

• Mass cytometry (CyTOF):

  • Label LENG9 antibodies with rare earth metals for highly multiplexed analysis

  • Enables simultaneous phenotyping of cells while quantifying LENG9 expression

  • Particularly valuable for heterogeneous tissues with multiple cell populations

• Targeted mass spectrometry:

  • Develop selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) assays

  • Use stable isotope-labeled peptide standards for absolute quantification

  • Provides orthogonal validation of antibody-based methods

Combining multiple quantification approaches provides the most robust assessment of LENG9 expression across different biological contexts.

What are the recommended approaches for studying LENG9 in non-human model organisms?

Studying LENG9 in non-human models requires careful consideration of evolutionary conservation and antibody cross-reactivity:

• Cross-reactivity assessment:

  • LENG9 orthologs have been identified in mouse, rat, bovine, frog, zebrafish, and chimpanzee models

  • Test human LENG9 antibodies against tissues from target species alongside human positive controls

  • Validate specificity in the non-human species using CRISPR/Cas9 knockout controls if possible

• Sequence homology analysis:

  • Align LENG9 protein sequences across species to identify conserved and divergent regions

  • Select antibodies targeting conserved epitopes for cross-species applications

  • Consider custom antibody development for highly divergent regions

• Model-specific considerations:

• Alternative approaches when antibodies fail:

  • CRISPR/Cas9 knock-in of epitope tags (FLAG, HA, etc.) to endogenous LENG9

  • Creation of fluorescent protein fusions for live imaging

  • Use of proximity labeling approaches (BioID, APEX) to study LENG9 interactomes

• Functional conservation assessment:

  • Compare LENG9 localization patterns across species using validated antibodies

  • Assess whether LENG9 interactions are conserved through co-immunoprecipitation

  • Determine if expression patterns during development are maintained across species

These approaches enable robust comparative studies of LENG9 biology across evolutionary diverse model systems.

How should researchers approach epitope mapping for LENG9 antibodies?

Epitope mapping for LENG9 antibodies requires systematic analysis to precisely identify antibody binding sites:

• Peptide array analysis:

  • Synthesize overlapping peptides (typically 15-20 amino acids with 5-10 amino acid overlaps) covering the entire LENG9 sequence

  • Immobilize peptides on membranes or glass slides

  • Probe with LENG9 antibodies and detect binding patterns

  • Identify peptides showing positive signals to narrow down epitope regions

• Recombinant fragment analysis:

  • Generate a series of truncated LENG9 constructs

  • Express and purify these fragments

  • Test antibody binding by Western blot or ELISA

  • Progressively narrow down the epitope region

• Alanine scanning mutagenesis:

  • Once a general epitope region is identified, create point mutations changing each amino acid to alanine

  • Test antibody binding to each mutant

  • Identify critical residues required for antibody recognition

• Hydrogen/deuterium exchange mass spectrometry:

  • Compare hydrogen/deuterium exchange rates of LENG9 alone versus LENG9-antibody complex

  • Regions protected from exchange in the complex represent potential epitopes

  • Provides structural insights about the antibody-antigen interaction

• X-ray crystallography or cryo-EM:

  • For highest resolution epitope mapping

  • Determine 3D structure of antibody-LENG9 complex

  • Identifies precise atomic interactions at the binding interface

• Computational approaches:

  • Use existing structural data or predicted models of LENG9

  • Apply epitope prediction algorithms to identify surface-exposed regions

  • Guide experimental approaches based on predictions

Epitope information helps explain cross-reactivity patterns, predict potential interference with protein function, and enables strategic selection of antibody pairs for sandwich assays.

What criteria should researchers use to evaluate and select LENG9 antibodies for specific applications?

Rigorous evaluation criteria ensure selection of optimal LENG9 antibodies for specific research applications:

• Validation documentation assessment:

  • Prioritize antibodies with knockout validation data

  • Evaluate the quality of validation data (e.g., clear Western blot bands at expected molecular weight, clean IHC staining)

  • Check for validation across multiple applications if the antibody will be used in different techniques

• Application-specific considerations:

• Antibody format evaluation:

  • Monoclonal vs. polyclonal: Monoclonals provide higher reproducibility; polyclonals may offer better sensitivity

  • Host species: Consider compatibility with other antibodies in multiplex applications

  • Clonality: For monoclonals, compare different clones recognizing distinct epitopes

  • Conjugation: Evaluate need for direct conjugates vs. secondary detection

• Reproducibility considerations:

  • Assess lot-to-lot consistency data if available

  • Check recombinant vs. immunized animal-derived antibodies (recombinant generally offers higher reproducibility)

  • Evaluate production scale and long-term availability for longitudinal studies

• Supporting literature evaluation:

  • Review publications using the antibody for similar applications

  • Assess quality of methods and controls in published studies

  • Contact authors for additional information if needed

These systematic evaluation criteria help researchers select antibodies most likely to yield reliable, reproducible results for their specific LENG9 studies.

How can researchers effectively compare the performance of different LENG9 antibodies?

Systematic comparison of LENG9 antibodies requires standardized evaluation protocols:

• Side-by-side testing framework:

  • Test all antibodies simultaneously on identical samples

  • Use consistent protocols for all technical parameters (sample preparation, blocking, detection)

  • Include appropriate positive and negative controls, especially LENG9 knockout samples

• Application-specific performance metrics:

For Western blot:

• Signal-to-noise ratio at equivalent concentrations

• Specificity (absence of non-specific bands)

• Sensitivity (detection limit using diluted samples)

• Reproducibility (consistency across replicate blots)

For IHC/IF:

• Background levels in negative control tissues

• Signal intensity in positive control tissues

• Subcellular localization precision

• Staining pattern consistency across tissue replicates

For immunoprecipitation:

• Capture efficiency (percentage of input LENG9 recovered)

• Purity of precipitated material (non-specific co-precipitants)

• Compatibility with downstream applications

• Quantitative scoring system:

  • Develop a weighted scoring rubric for each performance attribute

  • Assign numerical scores to each antibody across criteria

  • Calculate composite scores for objective comparison

• Blind evaluation:

  • Code antibodies to prevent bias during evaluation

  • Have multiple researchers independently assess performance

  • Compare scores and resolve discrepancies through consensus

This structured comparison approach enables objective selection of the best LENG9 antibody for specific research applications.

What documentation practices ensure reproducibility when using LENG9 antibodies in research?

Comprehensive documentation ensures experimental reproducibility and facilitates troubleshooting:

• Antibody metadata documentation:

  • Manufacturer and catalog number

  • Lot number (critical for reproducibility assessment)

  • Clone name/number for monoclonals

  • Host species and antibody isotype

  • Immunogen details (peptide sequence, fusion protein design)

  • Production method (hybridoma, recombinant, polyclonal)

• Validation documentation:

  • Specificity controls used (knockout, knockdown, peptide blocking)

  • Cross-reactivity testing results

  • Application-specific validation data

  • References to published validation studies

• Experimental protocol documentation:

  • Complete step-by-step procedures with exact buffer compositions

  • Antibody concentration and dilution calculation method

  • Incubation times and temperatures

  • Detection systems with complete details

  • Image acquisition parameters (exposure times, gain settings)

  • Quantification methods and software used

• Structured reporting format:

  • Laboratory notebooks with consistent antibody information sections

  • Electronic records with standardized fields for antibody details

  • Protocol management systems linking antibody information to methods

  • Sample tracking connected to antibody lot information

• Reporting standards for publication:

  • Follow journal-specific antibody reporting requirements

  • Include all validation data in supplements if not in main text

  • Provide RRID (Research Resource Identifier) for each antibody

  • Deposit detailed protocols in repositories like protocols.io

This comprehensive documentation approach ensures experimental reproducibility, facilitates troubleshooting, and enhances the scientific value of research using LENG9 antibodies.

How might LENG9 antibodies contribute to understanding tissue-specific expression patterns?

LENG9 antibodies can provide crucial insights into tissue-specific expression patterns through several advanced approaches:

• Multi-tissue profiling:

  • Apply validated LENG9 antibodies to tissue microarrays containing diverse normal and diseased tissues

  • Quantify expression levels and subcellular localization patterns across tissues

  • LENG9 shows notable expression in bronchus, vagina, and appendix tissues, suggesting specific biological roles in these contexts

• Single-cell resolution analysis:

  • Combine LENG9 antibodies with single-cell technologies:

    • Mass cytometry (CyTOF) with metal-conjugated LENG9 antibodies

    • Imaging mass cytometry for spatial context

    • CODEX multiplexed imaging for simultaneous detection of LENG9 and cell type markers

  • Correlate LENG9 protein expression with single-cell transcriptomics data

• Developmental trajectory mapping:

  • Apply LENG9 antibodies to tissue samples across developmental timepoints

  • Trace expression changes during tissue differentiation and maturation

  • Identify potential roles in tissue-specific developmental processes

• Pathological condition analysis:

  • Compare LENG9 expression between normal and diseased tissues

  • Evaluate expression changes in inflammatory conditions, cancer progression, or tissue remodeling

  • Assess potential as a diagnostic or prognostic biomarker

• Signaling context integration:

  • Combine LENG9 detection with phospho-specific antibodies against signaling molecules

  • Map LENG9 expression to specific signaling states in different tissues

  • Identify tissue-specific regulatory networks involving LENG9

These approaches could reveal previously unknown functions of LENG9 in specific tissue contexts and potentially identify new therapeutic targets or diagnostic markers.

What are the emerging trends in antibody validation that researchers should apply to LENG9 studies?

Emerging trends in antibody validation represent critical advances that should be applied to LENG9 research:

• Genetic knockout validation as the gold standard:

  • CRISPR/Cas9-mediated knockout of LENG9 in relevant cell lines provides definitive negative controls

  • Compare antibody performance between parent and knockout lines using multiple applications

  • This approach has been specifically highlighted as critical for avoiding misinterpretation of results

• Orthogonal validation methods:

  • Combine antibody-based detection with mass spectrometry

  • Compare protein levels detected by antibodies with targeted MS-based peptide quantification

  • Correlate with independent measures like LENG9 mRNA levels while accounting for post-transcriptional regulation

• Automated high-throughput validation pipelines:

  • Standardized testing across hundreds of conditions and fixation methods

  • Machine learning algorithms to assess staining patterns and predict specificity

  • Cloud-based repositories of validation data accessible to researchers

• Independent validation initiatives:

  • Multi-laboratory blind testing of antibody performance

  • Preregistered validation studies to prevent publication bias

  • Community-driven validation databases with standardized metrics

• Application-specific validation:

  • Recognition that antibody performance varies across applications

  • Validation data must match the intended research application

  • Development of application-specific validation standards and metrics

• Recombinant antibody technologies:

  • Shift toward sequence-defined recombinant antibodies for LENG9

  • Elimination of batch-to-batch variation inherent to hybridoma or serum-derived antibodies

  • Genetic engineering to optimize performance for specific applications

Implementing these emerging validation approaches for LENG9 antibodies will significantly enhance research reproducibility and reliability.

How can computational approaches enhance the interpretation of LENG9 antibody-based data?

Computational methods are increasingly essential for extracting maximum value from LENG9 antibody-based experiments:

• Image analysis algorithms:

  • Automated segmentation of cells/tissues in LENG9 immunofluorescence images

  • Quantification of expression levels across heterogeneous samples

  • Subcellular localization pattern recognition and classification

  • Correlation of LENG9 expression with morphological features

• Multi-omics data integration:

  • Computational frameworks linking LENG9 protein data with:

    • Transcriptomics (RNA-seq, single-cell RNA-seq)

    • Genomics (variant effects, regulatory elements)

    • Proteomics (interaction networks, post-translational modifications)

    • Metabolomics (pathway activities)

  • Bayesian networks to infer causal relationships

• Machine learning for pattern recognition:

  • Supervised learning to identify LENG9 expression patterns associated with biological states

  • Unsupervised learning to discover novel LENG9-related tissue or cell classifications

  • Deep learning approaches for feature extraction from complex tissue images

• Predictive modeling:

  • Structural modeling of LENG9 to predict antibody epitopes

  • Simulation of antibody-antigen interactions for optimized selection

  • In silico prediction of cross-reactivity risks

• Knowledge graph approaches:

  • Integration of LENG9 antibody data with published literature

  • Automated hypothesis generation through network analysis

  • Identification of knowledge gaps and prioritization of experiments

• Reproducibility enhancement tools:

  • Automated protocol generation based on antibody characteristics

  • Statistical power calculators specific to antibody-based experiments

  • Digital notebook systems capturing all experimental parameters

These computational approaches transform antibody-based data from descriptive observations into mechanistic insights and predictive models.