ENO3 Antibody

What is ENO3 and what cellular functions does it perform?

ENO3 (Enolase 3, beta, muscle) encodes the β-subunit of enolase, which is distributed across various tissues including liver, lung, skeletal muscle, and heart. This protein plays crucial roles in both glycogen and cholesterol metabolism . The functional protein has a calculated molecular weight of 47 kDa (434 amino acids) but is typically observed at 42-47 kDa in experimental conditions . ENO3 is particularly important in metabolic processes, as its deficiency has been linked to metabolic myopathies . Additionally, ENO3 accelerates hepatic cholesterol ester accumulation through the mediation of cholesteryl ester generation .

What applications are ENO3 antibodies best suited for?

ENO3 antibodies are versatile research tools validated for multiple applications including:

When selecting an ENO3 antibody, researchers should consider the specific experimental requirements and validate the antibody in their particular system, as optimal dilutions may vary based on the sample type and detection method employed .

How should I choose between monoclonal and polyclonal ENO3 antibodies?

The choice between monoclonal and polyclonal ENO3 antibodies depends on your experimental goals:

Monoclonal ENO3 Antibodies:

• Offer high specificity to a single epitope (e.g., mouse monoclonal IgG2b)

• Provide consistent lot-to-lot reproducibility

• Ideal for applications requiring high specificity and minimal background

• Available options include those reactive to specific regions (e.g., AA 228-277 or other defined sequences)

Polyclonal ENO3 Antibodies:

• Recognize multiple epitopes on the ENO3 protein

• Often provide stronger signals by binding multiple sites

• Typically generated in rabbits against recombinant fusion proteins

• Can be more forgiving in applications where protein conformation may vary

For novel research applications, testing both antibody types may help determine which provides optimal results for your specific experimental conditions.

What are the optimal storage conditions for ENO3 antibodies?

To maintain ENO3 antibody integrity and functionality, proper storage is essential:

• Store at -20°C for long-term preservation

• Antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Most are stable for one year after shipment when stored properly

• Avoid repeated freeze/thaw cycles that can degrade antibody quality

• For antibodies provided in small volumes (e.g., 20μl), some formulations contain 0.1% BSA for stabilization

• Aliquoting is generally recommended for antibodies without glycerol, but may be unnecessary for those in 50% glycerol storage buffer

Following these storage guidelines will help ensure experimental reproducibility and extend the usable life of your ENO3 antibodies.

What species reactivity should I consider when selecting an ENO3 antibody?

When selecting an ENO3 antibody, species cross-reactivity is a critical consideration:

For comparative studies across species, select antibodies with validated cross-reactivity for all target organisms. Western blot validation has confirmed ENO3 antibody detection in various samples including HeLa cells, HEK-293 cells, HepG2 cells, Jurkat cells, and skeletal muscle tissue from multiple species . Always perform preliminary validation tests when using antibodies in species not explicitly listed in the manufacturer's documentation.

How can I validate the specificity of ENO3 antibodies in my experimental system?

Rigorous antibody validation is essential for generating reliable research data. For ENO3 antibodies, consider this comprehensive validation approach:

• Positive and negative controls:

  • Use skeletal muscle tissue known to express high levels of ENO3 as a positive control

  • Include tissues with low/no ENO3 expression as negative controls

  • Consider using knockout/knockdown cell lines if available

• Cross-validation with multiple techniques:

  • Compare results from Western blot, immunofluorescence, and immunohistochemistry

  • Confirm the observed molecular weight matches the expected size (42-47 kDa)

  • For monoclonal antibodies, verify epitope-specific binding

• Peptide competition assay:

  • Pre-incubate the antibody with immunizing peptide

  • Compare signal reduction in samples with and without peptide blocking

• Orthogonal validation:

  • Correlate protein detection with mRNA expression data

  • Use mass spectrometry to confirm protein identity in immunoprecipitated samples

• Reproducibility assessment:

  • Test antibody performance across multiple batches of samples

  • Evaluate consistency across different lots of the antibody if possible

Document all validation steps thoroughly as supporting evidence for publications and maintain validated antibodies under optimal storage conditions to ensure consistent performance.

What are the considerations for using ENO3 antibodies in cancer research, particularly regarding the Warburg effect?

Recent research has implicated ENO3 in cancer metabolism, particularly through its relationship with the Warburg effect. When designing experiments to investigate this connection:

• Expression correlation studies:

  • Compare ENO3 levels in normal versus cancer tissues

  • Note that ENO3 expression patterns in tumors show contradictory results across different cancer types

  • Specifically examine STK11 mutant conditions where ENO3 up-regulation has been observed

• Metabolic pathway analysis:

  • Investigate ENO3's role in glycolytic flux using antibodies to track protein levels

  • Recent data establish that ENO3 promotes cell proliferation by enhancing the Warburg effect

  • Consider using ENO3 antibodies in conjunction with other glycolytic enzyme markers

• Clear cell renal cell carcinoma (ccRCC) studies:

  • ENO3's functional significance in the Warburg effect observed in ccRCC cells presents a specific research opportunity

  • Design experiments to clarify the still unclear expression and function of ENO3 in these cells

• Post-translational modification examination:

  • Investigate NSUN5-mediated m5C modification of ENO3, which has been implicated in its function

  • Use appropriate antibodies that can detect these modifications or employ immunoprecipitation followed by mass spectrometry

• Functional studies:

  • Combine antibody-based detection with knockdown/overexpression experiments

  • Assess changes in cancer cell metabolism, proliferation, and migration upon modulation of ENO3 levels

This methodological approach enables comprehensive investigation of ENO3's role in cancer metabolism while maximizing the utility of ENO3 antibodies in uncovering novel biological insights.

How can I optimize ENO3 antibody use for investigating metabolic myopathies?

ENO3 deficiency has been linked to metabolic myopathies , making it an important research target. To optimize antibody-based investigations:

• Tissue-specific analysis protocol:

  • Use skeletal muscle biopsies from patients with suspected metabolic myopathies

  • Compare with appropriate control samples from healthy individuals

  • Employ both frozen and paraffin-embedded tissue preparations for comprehensive analysis

• Staining optimization for muscle tissue:

  • For immunohistochemistry: Test antigen retrieval methods specific to muscle tissue

  • For immunofluorescence: Start with the recommended dilution range (1:50-1:200) and adjust based on signal-to-noise ratio

  • Include muscle-specific markers (e.g., desmin) for co-localization studies

• Quantitative analysis approaches:

  • Employ western blotting with gradient gels to resolve potential ENO3 isoforms

  • Consider using automated image analysis software for quantification

  • Normalize ENO3 expression to appropriate housekeeping proteins specific to muscle tissue

• Physiological correlation:

  • Correlate ENO3 antibody staining patterns with clinical parameters

  • Document glycogen accumulation using PAS staining in parallel sections

  • Assess mitochondrial function markers to understand the metabolic consequences

• Genetic correlation:

  • Combine ENO3 protein analysis with genotyping for known ENO3 mutations

  • Investigate protein expression patterns in carriers versus affected individuals

This methodological framework enables researchers to effectively use ENO3 antibodies when investigating the molecular basis of metabolic myopathies.

What are the recommended protocols for troubleshooting inconsistent results when using ENO3 antibodies?

When encountering inconsistent results with ENO3 antibodies, follow this systematic troubleshooting approach:

• Sample preparation issues:

  • Ensure complete protein denaturation for western blotting

  • Verify protein extraction efficiency from different tissue types

  • For skeletal muscle samples, pay special attention to myofibrillar protein extraction protocols

• Antibody-specific optimization:

  • Titrate antibody concentration systematically (WB: 1:5,000-1:50,000; IF/ICC: 1:200-1:800)

  • Test different blocking agents to reduce background

  • For polyclonal antibodies, consider lot-to-lot variation effects

• Detection system verification:

  • Evaluate secondary antibody specificity

  • For fluorescence-based detection, check for autofluorescence in muscle samples

  • Optimize exposure times or gain settings to prevent signal saturation

• Technical controls:

  • Include recombinant ENO3 protein as a positive control

  • Use loading controls appropriate for the experimental context

  • Perform parallel experiments with alternative antibodies targeting different ENO3 epitopes

• Documentation and standardization:

  • Record all experimental conditions in detail

  • Standardize protocols across experiments

  • Consider developing a laboratory-specific validated protocol once optimal conditions are determined

By systematically addressing these factors, researchers can improve the consistency and reliability of experiments using ENO3 antibodies.

How can ENO3 antibodies be utilized to study post-translational modifications?

Investigating post-translational modifications (PTMs) of ENO3 requires specialized approaches:

• Identification of potential PTM sites:

  • NSUN5-mediated m5C modification has been reported for ENO3

  • Review literature and databases for phosphorylation, acetylation, and other potential modifications

• Modification-specific detection strategies:

  • Use antibodies raised against specific ENO3 PTMs when available

  • Employ two-dimensional gel electrophoresis to separate modified forms

  • Consider enrichment strategies for phosphorylated or acetylated proteins

• Immunoprecipitation-based approaches:

  • Use ENO3 antibodies for immunoprecipitation followed by:

    • Western blotting with modification-specific antibodies

    • Mass spectrometry for comprehensive PTM profiling

    • Functional assays to determine effects on enzymatic activity

• Contextual analysis:

  • Compare PTM patterns across different physiological conditions

  • Investigate tissue-specific modification patterns

  • Assess changes in PTMs during disease progression, particularly in cancer models

• Functional correlation:

  • Correlate PTM changes with ENO3 enzymatic activity

  • Investigate how modifications affect protein-protein interactions

  • Determine if modifications alter subcellular localization

This methodological framework enables researchers to leverage ENO3 antibodies for detailed investigation of post-translational regulation mechanisms that may be critical in both normal physiology and disease states.