LECT2 Antibody, HRP conjugated

What is LECT2 and why is it significant for immunological research?

LECT2 (leukocyte cell-derived chemotaxin 2) is a 16.4 kilodalton protein that plays important roles in various immunological processes. It may also be known as chm-II, chm2, and chondromodulin-II in the literature . Recent studies have demonstrated LECT2's involvement in inflammatory diseases, particularly atopic dermatitis (AD), where it exacerbates disease progression by impairing skin barrier function and increasing inflammatory responses through the activation of the NF-κB signaling pathway . Understanding LECT2's molecular functions provides valuable insights into immune regulation and potential therapeutic targets for inflammatory conditions. The protein contains a zinc-binding domain critical for its stability, with zinc loss potentially contributing to protein misfolding and disease states .

What are the critical applications for LECT2 antibody, HRP conjugated in research settings?

LECT2 antibody, HRP conjugated serves multiple critical research applications:

The HRP conjugation eliminates the need for secondary antibodies, simplifying protocols and reducing background. Different suppliers offer varying reactivity profiles, with some antibodies detecting human LECT2 specifically, while others cross-react with mouse, rabbit, rat, or guinea pig LECT2 .

How should researchers select the appropriate LECT2 antibody, HRP conjugated for specific experimental needs?

Researchers should consider several factors when selecting a LECT2 antibody:

• Target epitope location: Antibodies targeting different regions (e.g., N-terminal vs. full-length) may yield different results, especially when studying LECT2 variants .

• Species reactivity: Ensure compatibility with your experimental model (human, mouse, etc.) .

• Validated applications: Confirm the antibody has been validated for your specific application (WB, IHC, ELISA) .

• Detection sensitivity: Consider the expected concentration of LECT2 in your samples.

• Lot-to-lot consistency: Request information on quality control processes from suppliers.

For studies involving the V40 and I40 variants of LECT2 associated with amyloidosis, careful antibody selection is particularly important to ensure detection specificity for the variant of interest .

What are the optimal conditions for Western blotting with LECT2 antibody, HRP conjugated?

Optimal Western blotting conditions for LECT2 detection require careful protocol optimization:

Critical considerations include ensuring complete protein transfer (particularly important for the 16.4 kDa LECT2), preventing non-specific binding, and implementing appropriate controls to validate specificity .

How can researchers effectively design experiments to study LECT2's role in atopic dermatitis using HRP-conjugated antibodies?

When designing experiments to study LECT2's role in atopic dermatitis, researchers should:

• Establish appropriate model systems:

  • In vitro: TNF-α/IFN-γ-induced HaCaT cells recapitulate key features of AD .

  • In vivo: DNCB-induced mouse models using both wild-type and LECT2 knockout mice .

• Implement comprehensive analytical approaches:

  • Analyze both tissue-specific mRNA expression (RT-qPCR) and systemic protein levels (ELISA) .

  • Evaluate barrier proteins (FLG, IVL, LOR) alongside inflammatory markers .

  • Assess NF-κB pathway activation via nuclear translocation studies .

• Design controlled interventions:

  • Compare LECT2 treatment effects in healthy and disease models .

  • Include appropriate dosing studies to establish dose-response relationships.

Recent research has demonstrated that LECT2 treatment increases inflammatory factor levels (TNF-α, IL-1β, IL-4, IL-6, IL-13, TSLP, RANTES) in TNF-α/IFN-γ-induced HaCaT cells, while decreasing barrier protein levels (FLG, IVL, LOR) . These molecular changes correlate with AD disease progression and severity.

What methodological approaches can be used to quantify LECT2 levels in complex biological samples?

Quantification of LECT2 in complex biological samples requires careful methodological consideration:

Laser microdissection coupled with mass spectrometry (LMD/MS) has emerged as a recommended method for confirming amyloid type in clinical specimens, including ALECT2 amyloidosis . For accurate quantification, researchers should include calibration curves using recombinant LECT2 standards and implement appropriate normalization strategies.

How does zinc binding affect LECT2 stability and detection using antibody-based methods?

Zinc binding significantly impacts LECT2 stability and detection in experimental systems:

• Stability implications:

  • Zinc-free LECT2 (apoLECT2) shows decreased stability and increased aggregation propensity .

  • Zinc affinity decreases approximately 60-fold when pH changes from 7.5 to 6.5, likely due to protonation of histidine residues at positions 35 and 120 .

  • Under normal blood pH (7.35-7.45), approximately 9-13% of LECT2 exists in the zinc-free form, increasing to 80% at pH 6.5 .

• Methodological considerations for detection:

  • Sample buffer composition should be carefully controlled for pH and metal chelators.

  • EDTA in sample buffers may remove zinc, potentially altering epitope accessibility.

  • Antibodies raised against different regions may have differential sensitivity to zinc-bound versus zinc-free LECT2.

• Experimental design implications:

  • Include both zinc-bound and zinc-free LECT2 controls when optimizing detection methods.

  • Consider pH effects when designing experiments, especially for kidney studies where pH can decrease to 5.5 in collecting ducts .

  • Account for potential competition from albumin, which chelates zinc in blood samples .

What are the critical considerations when studying the V40 and I40 variants of LECT2 using antibody-based detection methods?

When studying LECT2 variants, particularly the V40 and I40 variants associated with amyloidosis, researchers should consider:

• Antibody epitope specificity:

  • Verify whether your antibody can differentiate between variants or binds to a conserved region.

  • Consider using multiple antibodies targeting different epitopes for validation.

• Expression system selection:

  • Both E. coli and human embryonic kidney (HEK) cell expression systems have been successfully used for recombinant LECT2 variant production .

  • Mammalian expression systems may provide more physiologically relevant post-translational modifications.

• Stability and aggregation analysis:

  • Urea denaturation studies reveal similar stability for I40 and V40 variants .

  • Both variants show accelerated aggregation at pH 8 and after zinc removal .

  • Western blot detection may be affected by aggregation state; consider native versus denaturing conditions.

• Experimental controls:

  • Include both variant proteins as positive controls.

  • Implement knockdown/knockout controls to verify antibody specificity.

  • Consider the impact of buffer conditions on variant stability and detection.

How can researchers effectively design experiments to investigate LECT2 amyloidosis mechanisms?

Designing experiments to investigate LECT2 amyloidosis mechanisms requires a multifaceted approach:

• In vitro aggregation studies:

  • Compare aggregation kinetics of purified V40 and I40 LECT2 variants under physiologically relevant conditions .

  • Assess the impact of pH, temperature, and zinc concentration on aggregation propensity.

  • Utilize thioflavin T fluorescence assays to monitor amyloid formation in real-time.

• Structural analysis:

  • Employ circular dichroism (CD) spectroscopy to assess secondary structure changes during aggregation.

  • Use transmission electron microscopy (TEM) to characterize fibril morphology.

  • Consider X-ray crystallography or NMR for high-resolution structural differences between variants.

• Cellular models:

  • Develop cell lines expressing V40 or I40 LECT2 variants.

  • Assess intracellular aggregation using fluorescence microscopy.

  • Evaluate cellular responses to LECT2 aggregates (stress responses, inflammatory signaling).

• Tissue analysis:

  • Implement laser microdissection and mass spectrometry (LMD/MS) for analyzing clinical specimens .

  • Utilize co-immunostaining to assess co-localization with other amyloid components.

  • Quantify tissue distribution patterns of aggregates in affected organs.

What are common sources of technical variability when using LECT2 antibody, HRP conjugated, and how can they be addressed?

Technical variability in LECT2 antibody applications can arise from multiple sources:

To address these variables, implement a comprehensive quality control system including standardized positive and negative controls, consistent sample handling protocols, and regular antibody validation procedures.

How should researchers interpret contradictory results between different detection methods for LECT2?

When faced with contradictory results between detection methods:

• Consider method-specific limitations:

  • Western blot may detect denatured epitopes invisible to native-state methods.

  • ELISA may be affected by matrix effects absent in other techniques.

  • IHC results can be influenced by fixation methods that alter epitope accessibility.

• Implement sequential validation strategies:

  • Confirm findings with multiple antibody clones targeting different epitopes.

  • Supplement antibody-based detection with non-antibody methods (mass spectrometry).

  • Perform knockdown/knockout experiments to verify specificity.

• Evaluate the impact of LECT2 conformational states:

  • Zinc-bound versus zinc-free states may affect epitope accessibility .

  • Aggregated LECT2 may mask epitopes available in monomeric form.

  • Post-translational modifications may differ between experimental and biological samples.

• Consider biological variability:

  • Expression levels may genuinely differ between sample types.

  • Disease states may alter LECT2 conformation or localization.

  • Genetic variants (V40/I40) may affect detection efficiency by certain antibodies .

What emerging methodologies might enhance LECT2 research beyond current antibody-based approaches?

Several emerging technologies show promise for advancing LECT2 research:

• CRISPR-based technologies:

  • CRISPR activation/inhibition systems for precise modulation of LECT2 expression.

  • CRISPR knock-in models for studying LECT2 variants in physiologically relevant contexts.

  • CRISPR screens to identify functional interactors with LECT2.

• Advanced imaging techniques:

  • Super-resolution microscopy for visualizing LECT2 aggregation at nanoscale resolution.

  • Intravital microscopy for studying LECT2 dynamics in live animal models.

  • Correlative light and electron microscopy (CLEM) for combining functional and structural information.

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify proximal interactors of LECT2.

  • Split-BioID systems to study conditional interactions dependent on zinc binding.

• Systems biology approaches:

  • Multi-omics integration to understand LECT2's role in complex disease networks.

  • Mathematical modeling of LECT2 aggregation kinetics and pathway interactions.

  • Network analysis of LECT2-dependent signaling cascades in inflammatory contexts.

How might understanding LECT2's role in atopic dermatitis inform therapeutic strategies in other inflammatory conditions?

The elucidation of LECT2's role in atopic dermatitis provides several translational insights:

• Pathway-specific interventions:

  • LECT2's activation of the NF-κB signaling pathway in AD suggests potential therapeutic targets applicable to other inflammatory conditions .

  • The demonstrated effect on barrier proteins (FLG, IVL, LOR) may be relevant to other barrier dysfunction disorders .

• Biomarker development:

  • Serum LECT2 correlates with AD disease severity, suggesting utility as a biomarker in other inflammatory conditions .

  • The relationship between LECT2 levels and specific inflammatory cytokines (IL-4, IL-13, etc.) may inform cytokine-targeted therapies.

• Structural interventions:

  • Understanding the role of zinc in LECT2 stability suggests potential zinc supplementation strategies for conditions involving LECT2 dysfunction .

  • The differential stability of LECT2 variants provides insights for developing stabilizing compounds to prevent aggregation.

• Amyloidosis connections:

  • Mechanisms of LECT2 amyloidosis may share commonalities with other amyloid disorders, informing broader therapeutic approaches .

  • The Hispanic population's predisposition to ALECT2 amyloidosis highlights the importance of considering genetic diversity in inflammatory disease treatment strategies .