LEXM Antibody

What is LEXM and what is its biological significance?

LEXM is a protein encoded by the CIMAP2 gene, functioning as an immune regulator involved in modulating inflammatory responses and immune activation. The human version of LEXM has a canonical amino acid length of 418 residues and a protein mass of 47.6 kilodaltons, with two identified isoforms . Its role in regulating inflammation and immune processes makes it a valuable target for studying autoimmune disorders, chronic inflammatory conditions, and cancer . Understanding LEXM's function provides insights into fundamental immune system mechanisms and potential therapeutic approaches for immune-related diseases.

What types of LEXM antibodies are available for research purposes?

LEXM antibodies are available in multiple formats optimized for different research applications. These include:

• Unconjugated primary antibodies: Most commonly used for standard detection in Western blots, immunohistochemistry, and immunofluorescence applications .

• Conjugated antibodies: Available with various tags including:

  • Biotin-conjugated antibodies for enhanced sensitivity and signal amplification

  • HRP-conjugated antibodies optimized for ELISA and other enzymatic detection methods

• Polyclonal antibodies: Generated in rabbits against specific regions (e.g., middle region) or full-length proteins, offering broad epitope recognition .

The selection among these antibody types depends on experimental goals, required sensitivity, and specific detection methods.

How do I select the appropriate LEXM antibody for my research?

Selecting the optimal LEXM antibody requires consideration of several experimental factors:

• Target species: Ensure the antibody has confirmed reactivity against your species of interest. Available LEXM antibodies show varying reactivity profiles, with most demonstrating reactivity to human samples, while some also react with rat, mouse, rabbit, and other species .

• Application compatibility: Choose antibodies validated for your specific application. LEXM antibodies are validated for various applications with different recommended dilutions:

  • Western Blot: Typically 1:500-1:5000 dilution

  • ELISA: Usually 1:2000-1:10000 dilution

  • Immunohistochemistry: Generally 1:20-1:200 dilution

• Antibody format: Select between conjugated or unconjugated forms based on your detection system.

• Epitope recognition: Consider antibodies targeting specific regions if studying particular isoforms or domains of LEXM.

Thoroughly review product documentation and validation data before making your selection to ensure optimal performance in your experimental system.

What are the optimal protocols for using LEXM antibodies in Western blotting?

For optimal Western blot results with LEXM antibodies, follow these methodological guidelines:

• Sample preparation:

  • Extract proteins from tissues of interest (e.g., rat heart or skeletal muscle have shown positive results)

  • Use a buffer containing protease inhibitors to prevent degradation

  • Denature samples in loading buffer containing SDS and a reducing agent

• Gel electrophoresis and transfer:

  • Resolve proteins on a 10-12% SDS-PAGE gel (appropriate for ~47.6kDa LEXM protein)

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

• Antibody incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST

  • Incubate with primary LEXM antibody at 1:500-1:5000 dilution (optimized concentration will depend on specific antibody)

  • Use validated antibodies such as rabbit polyclonal antibodies that have demonstrated specificity in Western blot applications

  • Incubate with appropriate secondary antibody (e.g., Goat anti-rabbit IgG for rabbit-derived antibodies)

• Detection optimization:

  • For standard Western blots, a concentration of approximately 4μg/ml has been reported as effective

  • Expected band size is approximately 47.6 kDa, but confirm exact size based on your specific LEXM isoform

This protocol should be optimized for your specific experimental conditions and the particular LEXM antibody you are using.

How can I optimize immunohistochemistry protocols using LEXM antibodies?

For successful immunohistochemistry (IHC) detection of LEXM, follow these methodological recommendations:

• Sample preparation:

  • Fix tissue samples appropriately (4% paraformaldehyde is commonly used)

  • Process and embed tissues according to standard protocols

  • Section at 4-6μm thickness for optimal antibody penetration

• Antigen retrieval:

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 10-20 minutes) to balance antigen exposure and tissue preservation

• Antibody incubation and detection:

  • Block endogenous peroxidase with 3% hydrogen peroxide

  • Apply protein blocking solution to reduce background

  • Incubate with LEXM antibody at 1:20-1:200 dilution (specific optimal dilution depends on the antibody)

  • Use appropriate detection system (e.g., polymer-based detection systems for enhanced sensitivity)

  • Counterstain, dehydrate, and mount according to standard protocols

• Controls and validation:

  • Include positive control tissues known to express LEXM

  • Include negative controls (primary antibody omission or isotype control)

  • Validate specificity through comparative staining with different LEXM antibodies

These protocols should be optimized for each specific experimental condition, tissue type, and LEXM antibody to achieve optimal signal-to-noise ratio and specific staining.

What are the recommended protocols for ELISA using LEXM antibodies?

For developing effective ELISA assays with LEXM antibodies, follow these methodological steps:

• Plate preparation:

  • Coat high-binding ELISA plates with capture antibody or antigen

  • For direct ELISA, coat with LEXM-containing samples

  • For sandwich ELISA, coat with anti-LEXM capture antibody

• Sample preparation:

  • Prepare protein extracts from tissues or cell cultures

  • Dilute appropriately in coating buffer (typically carbonate-bicarbonate buffer, pH 9.6)

• Antibody incubation:

  • For direct ELISA: Apply LEXM antibody at 1:2000-1:10000 dilution

  • For sandwich ELISA: Use paired antibodies recognizing different epitopes

  • Biotin-conjugated or HRP-conjugated LEXM antibodies can enhance detection sensitivity

• Detection and analysis:

  • Develop using appropriate substrate (TMB for HRP-conjugated systems)

  • Read absorbance at appropriate wavelength

  • Calculate LEXM concentration using standard curve

• Optimization considerations:

  • Titrate antibody concentrations to determine optimal signal-to-noise ratio

  • Adjust blocking conditions to minimize background

  • Optimize incubation times and temperatures

Different LEXM antibody formats (non-conjugated, biotin-conjugated, HRP-conjugated) are available to accommodate various ELISA formats and detection systems .

How can I resolve poor signal or high background issues when using LEXM antibodies?

Common signal and background issues with LEXM antibodies can be systematically resolved through these methodological approaches:

For poor signal:

• Antibody concentration optimization:

  • Titrate antibody concentrations - try higher concentrations within the recommended range (e.g., 1:20 instead of 1:200 for IHC)

  • Ensure antibody storage conditions maintain activity (50% glycerol, 0.01M PBS, pH 7.4 at appropriate temperature)

• Antigen accessibility improvement:

  • Optimize antigen retrieval methods (try different buffers or longer retrieval times)

  • For membrane proteins, consider mild detergent addition during antibody incubation

  • Increase incubation time or temperature to improve antibody-antigen binding

• Detection system enhancement:

  • Switch to more sensitive detection systems (e.g., biotin-streptavidin amplification)

  • Ensure secondary antibodies are compatible with host species (e.g., anti-rabbit for rabbit polyclonal LEXM antibodies)

  • Verify detection reagents are functioning properly with positive controls

For high background:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time or concentration

  • Add blocking agents to antibody diluent

• Washing improvements:

  • Increase number and duration of wash steps

  • Use appropriate detergent concentration in wash buffers

  • Ensure complete removal of wash buffer between steps

• Antibody specificity verification:

  • Use highly purified antibodies (>95% protein G purified)

  • Perform antibody validation with positive and negative controls

  • Consider pre-absorption with blocking peptides if available

Each of these approaches should be tested systematically, changing one variable at a time to identify the optimal conditions for your specific experimental system.

How can I validate the specificity of LEXM antibodies in my experimental system?

Validating antibody specificity is crucial for reliable research results. For LEXM antibodies, implement these methodological validation strategies:

• Positive and negative control tissues/cells:

  • Use tissues known to express LEXM (e.g., rat heart tissue, rat skeletal muscle tissue have shown positive WB results)

  • Include tissues/cells where LEXM expression is absent or knocked down

  • Compare staining patterns with known cellular localization of LEXM

• Multiple antibody validation:

  • Test multiple LEXM antibodies targeting different epitopes

  • Compare results between antibodies from different suppliers or clones

  • Consistent results across antibodies increase confidence in specificity

• Molecular validation approaches:

  • Perform knockdown/knockout validation (siRNA, CRISPR) followed by antibody staining

  • Conduct overexpression studies and confirm increased signal

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

• Technical controls:

  • Include isotype controls to assess non-specific binding

  • Perform absorption controls with immunizing peptide if available

  • Test cross-reactivity with related proteins through bioinformatic analysis and experimental validation

• Correlation with alternative methods:

  • Confirm protein expression using orthogonal techniques (e.g., mRNA expression)

  • Compare antibody staining patterns with fluorescent protein tagging

Systematic implementation of these validation approaches provides confidence in antibody specificity and reliability of subsequent experimental results.

What are the optimal storage conditions and handling practices for maintaining LEXM antibody quality?

Proper storage and handling of LEXM antibodies is critical for maintaining their activity and specificity:

• Temperature considerations:

  • Store antibodies according to manufacturer recommendations (typically -20°C for long-term storage)

  • Avoid repeated freeze-thaw cycles by aliquoting upon receipt

  • For working stocks, short-term storage at 4°C (1-2 weeks) is generally acceptable

• Buffer composition importance:

  • LEXM antibodies are typically stored in preservative buffers containing:

    • 0.03% Proclin 300 as preservative

    • 50% Glycerol for stability

    • 0.01M PBS, pH 7.4 as buffer

  • Do not dilute stock antibodies unless preparing working dilutions

• Working dilution preparation:

  • Prepare fresh working dilutions before each experiment

  • Use appropriate diluent (typically PBS with 0.1-0.5% BSA or similar carrier protein)

  • Maintain aseptic technique when handling antibody solutions

• Contamination prevention:

  • Use sterile techniques when handling antibodies

  • Avoid introducing bacteria or fungi that could degrade antibodies

  • Consider adding sodium azide (0.02%) to working dilutions for short-term preservation

• Quality monitoring practices:

  • Document antibody performance over time

  • Include positive controls in each experiment to monitor antibody activity

  • If performance decreases, consider replacing with new antibody aliquot

Implementing these storage and handling practices will help maintain antibody quality and experimental consistency throughout your research project.

How can I design experiments to investigate LEXM's role in immune regulation and inflammation?

Designing robust experiments to elucidate LEXM's immune regulatory functions requires comprehensive methodological approaches:

• Expression correlation studies:

  • Analyze LEXM expression in various immune cell populations

  • Correlate LEXM expression with activation status using flow cytometry

  • Examine expression changes during inflammatory responses or immune challenges

  • Use validated LEXM antibodies for precise protein quantification in different immune contexts

• Functional perturbation approaches:

  • Design CRISPR/Cas9 knockout systems targeting LEXM

  • Develop siRNA or shRNA knockdown strategies

  • Create overexpression systems using lentiviral vectors

  • Assess impact on immune cell function using proliferation, cytokine production, and activation assays

• Protein interaction studies:

  • Perform co-immunoprecipitation with LEXM antibodies to identify binding partners

  • Conduct proximity ligation assays to confirm in situ interactions

  • Use mass spectrometry to characterize the LEXM interactome

  • Map interaction domains through truncation mutants

• Disease model investigations:

  • Examine LEXM expression in autoimmune disorder models

  • Assess impact of LEXM modulation on inflammatory disease progression

  • Correlate LEXM levels with disease severity and inflammatory markers

  • Study effects of targeting LEXM on disease outcomes

• Mechanistic pathway analysis:

  • Investigate LEXM's position in inflammatory signaling cascades

  • Examine effects on transcription factor activation (NF-κB, STATs)

  • Assess impact on cytokine and chemokine production networks

  • Determine effects on immune cell migration and tissue infiltration

These experimental approaches should be integrated to develop a comprehensive understanding of LEXM's role in immune regulation and inflammation, potentially revealing therapeutic opportunities for inflammatory diseases and cancer .

What methodologies can be employed to study LEXM in the context of ciliary microtubule function?

Given LEXM's association with ciliary microtubule associated protein 2 (CIMAP2) , these specialized methodologies can be employed to investigate its function:

• High-resolution imaging approaches:

  • Implement super-resolution microscopy (STORM, PALM, SIM) for detailed ciliary structure visualization

  • Use immunofluorescence with LEXM antibodies for precise localization within ciliary structures

  • Perform live-cell imaging with fluorescently tagged LEXM to track dynamics

  • Apply electron microscopy with immunogold labeling for ultrastructural localization

• Ciliary function assessment:

  • Measure ciliary beat frequency following LEXM perturbation

  • Analyze intraflagellar transport efficiency with and without LEXM

  • Assess ciliary length regulation and maintenance under varying LEXM levels

  • Evaluate signaling pathway activity dependent on ciliary function (e.g., Hedgehog signaling)

• Protein-protein interaction studies within ciliary context:

  • Identify ciliary-specific interaction partners through BioID or APEX proximity labeling

  • Perform co-immunoprecipitation with other ciliary proteins

  • Conduct yeast two-hybrid screening with ciliary protein libraries

  • Map functional interaction domains through truncation and point mutation analysis

• Microtubule dynamics investigations:

  • Analyze microtubule stability using depolymerization assays

  • Assess post-translational modifications of ciliary microtubules

  • Study microtubule nucleation and growth rates in presence/absence of LEXM

  • Examine recruitment of microtubule-associated proteins to ciliary axonemes

• Disease model applications:

  • Investigate LEXM function in ciliopathy models

  • Assess ciliary structure and function in tissues expressing mutant LEXM

  • Correlate LEXM expression with ciliary phenotypes in patient samples

  • Develop therapeutic approaches targeting LEXM for ciliopathies

These methodologies provide a framework for comprehensively understanding LEXM's role in ciliary microtubule function, potentially revealing novel insights into ciliopathies and related disorders.

What are the current limitations in LEXM antibody technology and how might they be addressed in future research?

Despite their utility, current LEXM antibody technologies face several limitations that future research should address:

• Isoform-specific detection challenges:

  • Current limitation: Most available antibodies cannot distinguish between the two identified LEXM isoforms

  • Future solutions:

    • Develop monoclonal antibodies targeting isoform-specific epitopes

    • Create recombinant antibodies with enhanced specificity

    • Implement epitope mapping to identify isoform-unique regions

    • Combine antibodies with mass spectrometry for isoform quantification

• Cross-reactivity concerns:

  • Current limitation: Potential cross-reactivity with related proteins limits absolute specificity

  • Future solutions:

    • Implement more rigorous validation in knockout/knockdown systems

    • Develop antibodies through subtractive immunization strategies

    • Create synthetic antibodies using phage display technology

    • Employ computational design for enhanced specificity

• Post-translational modification detection:

  • Current limitation: Existing antibodies typically cannot distinguish modified forms of LEXM

  • Future solutions:

    • Generate modification-specific antibodies (phospho-, ubiquitin-, etc.)

    • Develop proximity ligation assays for specific modified forms

    • Create biosensors sensitive to LEXM modifications

    • Combine antibody detection with mass spectrometry

• Quantification accuracy:

  • Current limitation: Variable binding affinities limit absolute quantification

  • Future solutions:

    • Develop calibrated standards for absolute quantification

    • Implement digital antibody-based detection methods

    • Create standardized reference materials for LEXM

    • Employ multiplex detection systems with internal controls

• Structural and functional epitope limitations:

  • Current limitation: Many antibodies may alter protein function upon binding

  • Future solutions:

    • Generate non-interfering nanobodies or aptamers

    • Map functional domains to design non-disruptive antibodies

    • Develop reversible binding reagents for dynamic studies

    • Create allosteric sensors that respond to but don't disrupt function

Addressing these limitations will significantly advance LEXM research capabilities, enabling more precise studies of its biological functions and potential therapeutic applications.