LENG1 Antibody

What is LENG1 protein and why is it studied in research?

LENG1 (leukocyte receptor cluster member 1) is a protein encoded by the LENG1 gene (Entrez Gene ID: 79165). While the precise function of this protein remains largely unknown, it is part of the leukocyte receptor cluster (LRC) and is studied in various contexts . Understanding LENG1's function requires reliable antibodies for detection and characterization in experimental settings. Currently, research is ongoing to elucidate its biological significance and potential role in cellular processes.

What types of LENG1 antibodies are available for research applications?

Multiple types of LENG1 antibodies are available for research, including:

The choice between polyclonal and monoclonal antibodies depends on your specific experimental requirements. Polyclonal antibodies recognize multiple epitopes on the target protein, potentially increasing sensitivity but with possible decreased specificity. Monoclonal antibodies recognize a single epitope, offering higher specificity but potentially lower sensitivity in certain applications .

What are the recommended applications for LENG1 antibodies?

Based on validation data, LENG1 antibodies are suitable for several research applications:

Always refer to the manufacturer's datasheet for specific recommendations, as optimal conditions may vary between antibody clones and experimental conditions .

How should I validate a LENG1 antibody for my specific application?

Antibody validation is critical for ensuring experimental reproducibility. For LENG1 antibodies, follow the five-pillar approach recommended by the scientific community:

• Genetic strategies (Gold standard): Use LENG1 knockout/knockdown cells as negative controls to confirm antibody specificity.

• Orthogonal strategies: Compare antibody results with an antibody-independent method (e.g., mass spectrometry, RNA expression).

• Independent antibody verification: Use multiple antibodies targeting different LENG1 epitopes and confirm similar results.

• Tagged-protein expression: Compare detection of overexpressed tagged LENG1 using both the antibody and tag-specific detection.

• Immunocapture mass spectrometry: For immunoprecipitation applications, confirm pulled-down proteins by mass spectrometry.

Document all validation steps thoroughly in your research methods section to enhance reproducibility .

What controls should I include when using LENG1 antibodies in my experiments?

Include these essential controls when working with LENG1 antibodies:

For LENG1 specifically, transfected cells overexpressing the protein provide excellent positive controls, as demonstrated in validation studies .

How can I determine the optimal working concentration for a LENG1 antibody?

Determine optimal working concentration through titration experiments:

• For Western blot:

  • Prepare a 2-fold dilution series (typically 1:250 to 1:2000)

  • Test against positive control samples

  • Select concentration with optimal signal-to-noise ratio

  • Document exposure times for reproducibility

• For immunofluorescence:

  • Test dilutions ranging from 1:50 to 1:500

  • Include negative controls at each dilution

  • Assess background levels and specific signal intensity

  • Consider counterstaining with subcellular markers

• For flow cytometry:

  • Prepare 3-5 different concentrations

  • Calculate signal-to-noise ratio at each concentration

  • Use titration curves to identify saturation point

  • Select concentration below saturation with acceptable signal

Document optimization experiments thoroughly in laboratory records .

How can I use LENG1 antibodies in co-immunoprecipitation to identify protein interaction partners?

For co-immunoprecipitation (co-IP) with LENG1 antibodies:

• Preparation:

  • Use freshly prepared cell/tissue lysates

  • Select lysis buffer that preserves protein interactions

  • Pre-clear lysates with protein A/G beads

• Immunoprecipitation:

  • Use 2-5 μg antibody per 500 μg protein lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add protein A/G magnetic beads (typically 50 μl)

  • Wash stringently (at least 3-5 times)

• Controls:

  • Input control (5-10% of lysate)

  • IgG control (same species/isotype as LENG1 antibody)

  • Reverse co-IP (if antibody to suspected interactor is available)

• Analysis:

  • Western blot for suspected interaction partners

  • Mass spectrometry for unbiased discovery approach

For LENG1, protein interaction studies are particularly valuable given its relatively unknown function. Use cross-linking approaches if interactions appear weak or transient .

What are the considerations for using LENG1 antibodies in immunohistochemistry applications?

When using LENG1 antibodies for immunohistochemistry:

• Tissue preparation considerations:

  • Fixation method significantly impacts epitope availability

  • Test both formalin-fixed paraffin-embedded (FFPE) and frozen sections

  • Optimize antigen retrieval methods (heat-induced vs. enzymatic)

• Validation steps:

  • Use orthogonal validation by correlating with RNA expression in tissues

  • Compare staining patterns between independent LENG1 antibodies

  • Include positive and negative control tissues

• Signal amplification:

  • Consider tyramide signal amplification for low abundance targets

  • Balance sensitivity needs with background concerns

  • Document all amplification steps precisely

• Quantification approaches:

  • Develop clear scoring criteria for staining intensity

  • Use digital image analysis when possible

  • Report both staining intensity and percentage of positive cells

Since LENG1's function remains unclear, correlating its expression with tissue-specific markers can provide functional insights .

How can I measure the affinity and specificity of LENG1 antibodies quantitatively?

Quantitative assessment of LENG1 antibody performance:

• Surface Plasmon Resonance (SPR):

  • Determine binding kinetics (kon, koff)

  • Calculate equilibrium dissociation constant (KD)

  • Compare binding to LENG1 vs. potential cross-reactive proteins

  • Typical high-quality research antibodies show KD in nM range

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Develop a quantitative ELISA using recombinant LENG1

  • Generate standard curves at multiple antibody concentrations

  • Calculate EC50 values as measure of binding strength

  • Test cross-reactivity with structurally similar proteins

• Biolayer Interferometry:

  • Measure real-time binding without labeling requirements

  • Determine binding kinetics in different buffer conditions

  • Assess epitope binning with multiple LENG1 antibodies

• Competitive Binding Assays:

  • Determine selectivity through competition experiments

  • Calculate IC50 values for direct comparison

These quantitative measurements provide objective quality assessments beyond simple positive/negative results .

What are common issues with LENG1 antibody performance and how can they be resolved?

Common LENG1 antibody challenges and solutions:

For LENG1 specifically, be aware that different isoforms may exist, potentially complicating band pattern interpretation in Western blots .

How do I optimize LENG1 antibody conditions for flow cytometry applications?

Optimization strategy for flow cytometry:

• Sample preparation:

  • Determine optimal fixation/permeabilization method

  • Test multiple permeabilization reagents if LENG1 detection is intracellular

  • Titrate cell concentration (typically 1×10^6 cells/100 μl)

• Staining protocol optimization:

  • Test different antibody concentrations (1:50 to 1:200 dilution range)

  • Optimize incubation time and temperature

  • Evaluate different blocking solutions to reduce background

  • Consider sequential staining for multiparameter analysis

• Fluorochrome selection:

  • Choose fluorochromes based on expected expression level

  • Brighter fluorochromes (PE, APC) for lower expression targets

  • Perform fluorescence-minus-one (FMO) controls

  • Include compensation controls for multicolor panels

• Data analysis:

  • Use appropriate gating strategies

  • Compare median fluorescence intensity (MFI)

  • Calculate staining index: (MFIsample - MFIcontrol)/2 × SDcontrol

Document all optimization steps in standard operating procedures for reproducibility .

What approaches can help resolve cross-reactivity issues with LENG1 antibodies?

Addressing cross-reactivity problems:

• Rigorous validation:

  • Implement genetic knockout controls (CRISPR/Cas9)

  • Use siRNA knockdown if knockout is not feasible

  • Compare results across multiple LENG1 antibodies targeting different epitopes

• Experimental modifications:

  • Increase blocking stringency (BSA, serum, commercial blockers)

  • Add detergents to reduce hydrophobic interactions

  • Adjust salt concentration in buffers

  • Reduce primary antibody concentration

• Advanced approaches:

  • Pre-absorb antibody with recombinant cross-reactive proteins

  • Perform epitope mapping to identify cross-reactive regions

  • Consider using monoclonal antibodies with defined epitopes

  • Explore recombinant antibody technology for greater specificity

• Documentation:

  • Report all cross-reactivity observed

  • Provide molecular weights of non-specific bands

  • Document how cross-reactivity was addressed

Cross-reactivity assessment is particularly important for lesser-studied proteins like LENG1, where complete characterization is still evolving .

How should I interpret contradictory results between different LENG1 antibodies?

When facing contradictory results:

• Systematic assessment:

  • Compare antibody properties (polyclonal vs. monoclonal, epitope locations)

  • Evaluate validation evidence for each antibody

  • Review experimental conditions for each experiment

• Resolution approaches:

  • Perform side-by-side comparison with identical samples

  • Validate with orthogonal methods (RNA expression, mass spectrometry)

  • Implement genetic controls (knockdown/knockout)

  • Consider isoform-specific detection differences

• Interpretation framework:

  • Multiple epitope-validated antibodies showing different results may suggest:

    • Protein isoforms with different epitope accessibility

    • Post-translational modifications affecting epitope recognition

    • Context-dependent protein conformations

    • Technical issues with one antibody

• Reporting guidelines:

  • Document all contradictions transparently

  • Report antibody catalog numbers, lots, and validation methods

  • Consider publishing contradictory findings to advance field knowledge

Such contradictions can actually provide valuable insights into protein biology when thoroughly investigated .

What quantitative methods should I use to analyze LENG1 expression levels in different experimental conditions?

Quantitative analysis approaches:

• Western blot quantification:

  • Use digital image acquisition with linear dynamic range

  • Perform densitometry with background subtraction

  • Normalize to loading controls (GAPDH, β-actin)

  • Generate standard curves with recombinant protein (absolute quantification)

  • Report relative fold-changes for comparative studies

• Immunofluorescence quantification:

  • Use consistent exposure settings across all samples

  • Measure mean fluorescence intensity within defined regions

  • Count positive cells using consistent threshold criteria

  • Apply automated image analysis software for unbiased assessment

• Flow cytometry quantification:

  • Report median fluorescence intensity (MFI)

  • Calculate percent positive using proper gating strategies

  • Consider using quantitative beads for absolute molecule quantification

  • Apply statistical methods appropriate for flow data

• Statistical analysis:

  • Use appropriate statistical tests based on data distribution

  • Report biological and technical replicates separately

  • Consider sample size calculations for adequate power

  • Report confidence intervals along with p-values

What minimum reporting standards should I follow when publishing research using LENG1 antibodies?

Comprehensive reporting standards:

• Antibody identification information:

  • Manufacturer and catalog number

  • Clone identity (for monoclonals)

  • Lot number (especially for polyclonals)

  • RRID (Research Resource Identifier) when available

• Validation evidence:

  • Methods used to validate specificity

  • Controls included in experiments

  • Supporting data from orthogonal methods

  • References to previous validation studies

• Experimental conditions:

  • Detailed protocol including buffer compositions

  • Antibody dilutions and incubation conditions

  • Sample preparation methods

  • Image acquisition parameters

• Quantification methods:

  • Software used for analysis

  • Normalization approaches

  • Statistical methods applied

  • Raw data availability statement

These standards align with the growing movement toward improved research reproducibility in antibody-based studies .

How can machine learning and computational approaches improve LENG1 antibody selection and validation?

Emerging computational approaches:

• Epitope prediction algorithms:

  • Use structure prediction tools to identify accessible LENG1 epitopes

  • Apply sequence conservation analysis to select epitopes in conserved/variable regions

  • Implement immunogenicity prediction for antibody development

  • Consider B-cell epitope prediction algorithms to identify potential binding sites

• Machine learning for validation:

  • Develop models to predict antibody specificity from validation data

  • Use image analysis algorithms to quantify staining patterns

  • Apply natural language processing to mine existing literature for validation evidence

  • Develop prediction tools for cross-reactivity based on sequence similarity

• Deep learning antibody generation:

  • Recent research demonstrates the ability to computationally generate antibody sequences with desirable properties

  • In-silico antibody libraries can be screened for theoretical binding to LENG1

  • Computationally designed antibodies show high expression, monomer content, and thermal stability

• Integration with structural biology:

  • Use AlphaFold2 or similar tools to predict LENG1 structure

  • Model antibody-antigen interactions in silico

  • Prioritize antibodies targeting structurally well-defined epitopes

These computational approaches can complement experimental validation to enhance antibody selection efficiency .

What considerations are important when studying LENG1 in disease models or clinical samples?

Translational research considerations:

• Disease context relevance:

  • Research the potential role of LENG1 in specific disease contexts

  • Consider how disease states might alter protein localization or modification

  • Validate antibodies in relevant disease models before clinical samples

  • Be aware that altered protein expression may affect epitope accessibility

• Sample-specific validation:

  • Validate antibodies specifically in each sample type (tissue, cell type)

  • Consider fixation and processing effects on epitope preservation

  • Include appropriate disease and normal controls

  • Be aware of potential cross-reactivity with disease-associated proteins

• Quantification challenges:

  • Develop standardized scoring systems for tissue analysis

  • Consider digital pathology approaches for objective quantification

  • Use multiple antibodies targeting different epitopes when possible

  • Include technical and biological replicates

• Translation to diagnostics:

  • Validate extensively before diagnostic consideration

  • Assess reproducibility across multiple laboratories

  • Consider automated detection systems to reduce subjectivity

  • Document all validation steps comprehensively

These considerations are particularly important as research moves from basic to translational applications .

How do post-translational modifications affect LENG1 antibody recognition, and how can I study these modifications?

Post-translational modification considerations:

• Impact on antibody binding:

  • Phosphorylation, glycosylation, or other modifications may block epitopes

  • Some antibodies may preferentially recognize modified forms

  • Modification patterns may vary by cell type or condition

  • Different results between antibodies may reflect modification detection differences

• Modification-specific studies:

  • Use modification-specific antibodies when available

  • Implement enzymatic treatments to remove specific modifications

  • Apply phosphatase treatment to study phosphorylation effects

  • Consider mass spectrometry to map modification sites

• Comprehensive characterization:

  • Combine immunoprecipitation with mass spectrometry

  • Use 2D gel electrophoresis to separate modified forms

  • Apply Phos-tag gels for phosphorylation studies

  • Consider site-directed mutagenesis of potential modification sites

• Reporting frameworks:

  • Document specific modification state detected by each antibody

  • Report modification-dependent changes in recognition patterns

  • Consider whether modifications affect protein function or localization

  • Validate modification findings with orthogonal approaches

Understanding post-translational modifications may provide key insights into LENG1 function and regulation .