ELOVL2 Antibody

What is ELOVL2 and what is its cellular function in lipid metabolism?

ELOVL2 is a transmembrane protein that catalyzes the first and rate-limiting reaction of the four reactions that constitute the long-chain fatty acids elongation cycle. It functions specifically in the synthesis of polyunsaturated very long chain fatty acids (C20- and C22-PUFA), with the highest activity toward C20:4(n-6) acyl-CoA substrates. ELOVL2 is primarily localized to the endoplasmic reticulum membrane where it participates in the production of polyunsaturated VLCFAs that serve as precursors for membrane lipids and lipid mediators . Its enzymatic activity is particularly important for converting docosapentaenoic acid (DPA, 22:5n-3) to 24:5n-3, which can lead to the formation of docosahexaenoic acid (DHA), a fatty acid crucial for retinal and brain function .

How is ELOVL2 expression regulated at the epigenetic level?

ELOVL2 expression is predominantly regulated through DNA methylation of its promoter region. Multiple studies have established that the methylation state of the ELOVL2 promoter is one of the most robust biomarkers of human age. Research demonstrates an age-related inverse relationship where increased methylation of the promoter corresponds with decreased ELOVL2 expression. This epigenetic regulation has been observed across multiple tissues including blood, retina, and liver . In aging cell models (WI38 and IMR90), higher cell population doubling numbers correlate with increased ELOVL2 promoter methylation and decreased gene expression, further supporting its role as a molecular marker of aging .

What are the key experimental models available for ELOVL2 research?

Researchers investigating ELOVL2 function employ various experimental systems:

These models enable comprehensive investigation of ELOVL2's roles in lipid metabolism, aging processes, and disease pathogenesis across different biological contexts .

What criteria should guide selection of an appropriate ELOVL2 antibody for specific experimental applications?

When selecting an ELOVL2 antibody, researchers should consider multiple parameters to ensure experimental success:

• Target specificity: Antibodies targeting different epitopes (e.g., N-terminal region amino acids 1-27) may have different detection capabilities .

• Species reactivity: Confirm that the antibody recognizes ELOVL2 in your experimental species. Many antibodies are validated for human samples with predicted reactivity in mouse or other species .

• Application compatibility: Verify the antibody has been validated for your intended application (WB, IHC-P, FC, etc.) with appropriate dilution recommendations .

• Clonality and host: Consider whether polyclonal (broader epitope recognition) or monoclonal (higher specificity) antibodies best suit your experimental needs .

• Detection method compatibility: Ensure the antibody is compatible with your visualization method (fluorescence, enzymatic, etc.) .

What validation protocols should be implemented to confirm ELOVL2 antibody specificity?

A comprehensive validation strategy should include:

• Positive and negative controls: Use tissues/cells with known ELOVL2 expression levels (e.g., liver as positive control) .

• Molecular weight verification: Confirm detection at the expected molecular weight of approximately 35 kDa in Western blot applications .

• Knockdown/knockout validation: Compare antibody signal in wild-type versus ELOVL2-depleted samples generated through CRISPR/Cas9 or shRNA approaches .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal specificity.

• Cross-validation: Compare results using multiple antibodies targeting different ELOVL2 epitopes to increase confidence in specificity.

• Cross-species reactivity testing: Verify whether predicted cross-reactivity with other species (zebrafish, horse, rabbit, dog, Xenopus) is accurate if relevant to your research .

What are the optimized protocols for ELOVL2 detection in different experimental contexts?

For Western blot applications:

• Sample preparation: Standard protein extraction protocols with particular attention to membrane protein solubilization

• Recommended dilution: 1:1000 for most ELOVL2 antibodies

• Expected molecular weight: 35 kDa

• Detection system: Standard secondary antibody approaches

For immunohistochemistry (IHC-P):

• Recommended dilution: 1:50-1:100

• Antigen retrieval: Heat-mediated retrieval recommended for formalin-fixed tissues

• Visualization: DAB or fluorescent secondary antibodies depending on experimental needs

For in situ hybridization (alternative to antibody-based detection):

• RNAscope probes can effectively detect ELOVL2 mRNA in specific cell types

• In mouse retina, ELOVL2 expression is predominantly observed in the photoreceptor layer (particularly cones) and retinal pigment epithelium (RPE)

How are ELOVL2 antibodies utilized to investigate the relationship between ELOVL2 methylation and aging?

ELOVL2 antibodies serve as essential tools in age-related methylation research through multiple approaches:

• Correlation studies: Researchers use ELOVL2 antibodies in combination with methylation analysis to correlate protein expression levels with promoter methylation status across different age groups. This typically involves:

  • Methylated DNA immunoprecipitation (MeDIP) or bisulfite sequencing to assess methylation levels

  • Western blot or immunohistochemistry with ELOVL2 antibodies to quantify protein expression

  • Statistical analysis to establish correlation between methylation and expression

• Intervention studies: ELOVL2 antibodies can evaluate the effectiveness of demethylating agents like 5-Aza-2'-deoxycytidine (5-Aza-dc) in restoring ELOVL2 expression. These experiments have demonstrated that reversal of promoter hypermethylation through intravitreal injection of 5-Aza-dc leads to increased ELOVL2 expression and rescue of age-related decline in visual function .

• Tissue-specific aging research: ELOVL2 antibodies have revealed that age-related decreases in ELOVL2 expression occur in multiple tissues including retina and liver, with corresponding increases in promoter methylation .

What is the methodological approach for studying ELOVL2's role in cancer progression using antibodies?

Investigating ELOVL2's oncogenic functions requires a multifaceted approach where antibodies play a central role:

• Expression analysis in tumor tissues: Immunohistochemistry using ELOVL2 antibodies has revealed elevated ELOVL2 expression in various renal cell carcinoma subtypes (ccRCC, pRCC, chRCC), with higher expression correlating with poor prognosis .

• Functional studies using gene editing: Following CRISPR/Cas9-mediated knockdown of ELOVL2, antibodies can monitor the efficiency of protein depletion and subsequent effects on:

  • Fatty acid elongation

  • Lipid droplet production

  • Cellular proliferation

  • Apoptosis induction

• Mechanistic investigations: ELOVL2 antibodies help elucidate how ELOVL2 depletion affects endoplasmic reticulum homeostasis, potentially explaining the mechanism by which ELOVL2 knockdown promotes apoptosis in cancer cells .

• In vivo tumor growth studies: ELOVL2 antibodies can assess protein expression in xenograft tumors, correlating expression levels with tumor growth rates .

How can researchers quantitatively assess ELOVL2 expression changes in response to experimental manipulations?

Quantitative assessment of ELOVL2 expression requires rigorous methodological approaches:

• Western blot densitometry:

  • Standardize protein loading with appropriate housekeeping controls

  • Use calibration curves with recombinant ELOVL2 standards if absolute quantification is needed

  • Apply digital image analysis software for densitometric quantification

  • Normalize ELOVL2 signal to loading controls

• Quantitative immunohistochemistry:

  • Use standardized staining protocols with consistent antibody dilutions

  • Employ digital pathology tools to quantify staining intensity

  • Include appropriate positive and negative controls on each slide

  • Consider automated image analysis for unbiased quantification

• Flow cytometry:

  • Optimize permeabilization protocols for this transmembrane protein

  • Use appropriate isotype controls

  • Consider dual staining with organelle markers to confirm subcellular localization

  • Report results as mean fluorescence intensity (MFI) or percent positive cells

What mechanisms link ELOVL2 dysfunction to age-related macular degeneration?

ELOVL2 dysfunction contributes to age-related macular degeneration (AMD) pathogenesis through several interconnected mechanisms:

• Disruption of DHA synthesis: ELOVL2 is critical for converting docosapentaenoic acid (DPA) to docosahexaenoic acid (DHA), which is the main polyunsaturated fatty acid in the retina. DHA promotes healthy retinal function and protects against oxidative stress and light damage .

• Formation of sub-RPE deposits: Mice with the C234W point mutation that disrupts ELOVL2-specific enzymatic activity develop deposits underneath the retinal pigment epithelium containing components found in human drusen, a pathologic hallmark of AMD .

• Premature visual decline: ELOVL2 mutant mice show electrophysiological characteristics of accelerated visual aging and early appearance of autofluorescent deposits, established markers of retinal aging .

• Epigenetic regulation: Age-related hypermethylation of the ELOVL2 promoter decreases its expression, potentially contributing to AMD susceptibility. Experimental reversal of this hypermethylation rescues age-related decline in visual function .

These findings establish ELOVL2 as a critical regulator of retinal aging and suggest potential therapeutic strategies for AMD treatment through modulation of ELOVL2 activity or expression.

How do ELOVL2 antibodies contribute to understanding cancer metabolism in renal cell carcinoma?

ELOVL2 antibodies have revealed critical insights into cancer metabolism in renal cell carcinoma:

• Expression profiling: ELOVL2 antibodies have demonstrated elevated ELOVL2 expression in multiple RCC subtypes, with higher expression correlating with poor prognosis .

• Metabolic pathway alterations: Following ELOVL2 knockdown, antibody-based detection methods have shown:

  • Suppression of long-chain polyunsaturated FA elongation

  • Increased lipid droplet production in renal cancer cells

  • Altered endoplasmic reticulum homeostasis

• Cellular effects: ELOVL2 antibodies have helped establish that ELOVL2 ablation suppresses cellular proliferation through induction of apoptosis in vitro and attenuates tumor growth in vivo .

• Therapeutic potential: These findings suggest ELOVL2 may represent an attractive novel target for RCC therapy, with antibodies serving as valuable tools for validating therapeutic approaches targeting this enzyme .

What experimental approaches combining genetic and antibody-based methods best elucidate ELOVL2's role in aging?

Comprehensive investigation of ELOVL2's role in aging benefits from integrated methodological approaches:

• Genetic manipulation with antibody-based validation:

  • CRISPR/Cas9-mediated gene editing to create specific ELOVL2 mutations (e.g., C234W)

  • Antibody confirmation of protein expression despite functional impairment

  • Correlation of phenotypic changes with protein levels

• Epigenetic modulation with expression monitoring:

  • Treatment with demethylating agents like 5-Aza-2'-deoxycytidine

  • Antibody-based quantification of restored ELOVL2 expression

  • Functional assessment of age-related improvements

• Age-related expression profiling:

  • Cross-sectional or longitudinal sampling across age groups

  • Antibody detection of ELOVL2 in multiple tissues

  • Correlation with methylation status and functional parameters

• Cell models of aging:

  • WI38 and IMR90 cell lines at different population doublings

  • Antibody quantification of ELOVL2 expression changes

  • Correlation with cellular senescence markers

These integrated approaches have established ELOVL2 as a molecular regulator of aging with potential therapeutic implications for age-related diseases.

What are promising therapeutic strategies targeting ELOVL2 that would require antibody-based validation?

Several therapeutic approaches targeting ELOVL2 show promise and would require antibody-based validation:

• Epigenetic modulation:

  • DNA demethylating agents like 5-Aza-2'-deoxycytidine could reverse age-related ELOVL2 hypermethylation

  • ELOVL2 antibodies would be essential to confirm restored protein expression

  • Tissue-specific delivery systems (e.g., intravitreal injection for retinal applications) would need validation

• Gene therapy approaches:

  • Viral vector-mediated ELOVL2 overexpression in affected tissues

  • Antibodies would confirm successful transgene expression

  • Long-term expression stability would require antibody-based monitoring

• Small molecule modulators:

  • Compounds enhancing residual ELOVL2 activity

  • Antibody-based assays to screen compound libraries

  • Confirmation of downstream metabolic effects

• Cancer-targeted therapies:

  • ELOVL2 inhibitors for cancers with ELOVL2 overexpression

  • Antibodies to monitor target engagement and expression changes

  • Combination therapies affecting lipid metabolism pathways

What methodological challenges remain in studying ELOVL2 function and how might they be addressed?

Several technical challenges complicate ELOVL2 research:

• Membrane protein detection limitations:

  • Challenge: As an ER membrane protein, ELOVL2 can be difficult to extract and detect

  • Solution: Optimize membrane protein extraction protocols; use epitope tags for difficult-to-detect regions

• Distinguishing between ELOVL family members:

  • Challenge: The ELOVL family includes seven members with overlapping functions

  • Solution: Develop highly specific antibodies targeting unique regions; validate with knockout controls

• Tissue-specific expression analysis:

  • Challenge: ELOVL2 expression varies across tissues and cell types

  • Solution: Single-cell approaches combining in situ hybridization with immunofluorescence

• Functional activity assessment:

  • Challenge: Protein expression may not directly correlate with enzymatic activity

  • Solution: Couple antibody detection with lipidomic analysis of specific fatty acid products

• Temporal dynamics of regulation:

  • Challenge: Age-related changes occur gradually over long timeframes

  • Solution: Develop conditional expression systems with antibody-based validation in model organisms

How might ELOVL2 research integrate with broader studies of lipid metabolism in disease and aging?

ELOVL2 research represents a critical node in the broader understanding of lipid metabolism in disease and aging:

• Integration with lipidomic profiling:

  • Correlate ELOVL2 expression (detected via antibodies) with comprehensive lipid profiles

  • Identify specific fatty acid species most affected by ELOVL2 dysfunction

  • Map changes to broader metabolic networks

• Cross-disease comparisons:

  • Apply consistent antibody-based methodologies across multiple disease models

  • Identify common and distinct roles of ELOVL2 in cancer, neurodegenerative diseases, and age-related pathologies

  • Develop disease-specific intervention strategies

• Multi-omics approaches:

  • Combine antibody-detected protein expression with transcriptomics, epigenomics, and metabolomics

  • Create integrated models of ELOVL2 regulation and function

  • Identify key regulatory nodes for therapeutic targeting

• Translational biomarker development:

  • Validate ELOVL2 methylation as an aging biomarker across populations

  • Correlate methylation patterns with protein expression in accessible tissues

  • Develop minimally invasive methods to monitor ELOVL2-related aging processes

Through these integrated approaches, ELOVL2 research promises to enhance our understanding of lipid metabolism in aging and disease while identifying novel therapeutic targets and biomarkers.