ELOVL6 Antibody

What is ELOVL6 and what is its primary function in cellular metabolism?

ELOVL6, sometimes also known as LCE (Long Chain Fatty acid Elongase), functions as an enzyme involved in the elongation of long-chain fatty acids . It catalyzes the first and rate-limiting reaction of the four reactions that constitute the long-chain fatty acids elongation cycle, allowing the addition of 2 carbons to the chain of long- and very long-chain fatty acids (VLCFAs) per cycle . This endoplasmic reticulum-bound enzyme primarily elongates saturated and monounsaturated fatty acids with 12, 14, and 16 carbons, showing higher activity toward C16:0 acyl-CoAs .

ELOVL6 has significant physiological importance as it may participate in the production of saturated and monounsaturated VLCFAs of different chain lengths that serve as precursors of membrane lipids and lipid mediators involved in multiple biological processes .

What antibody applications are recommended for ELOVL6 detection?

Based on current research and commercial availability, ELOVL6 antibodies have been validated for several applications:

For immunohistochemistry applications, researchers should consider antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 for optimal results . The recommended dilution for IHC applications ranges from 1:50 to 1:500, though researchers should titrate the antibody in their specific testing system to obtain optimal results .

What positive controls are recommended when validating ELOVL6 antibodies?

When validating ELOVL6 antibodies, several positive controls have been established in the literature:

For tissue samples, human hepatocirrhosis tissue and human brain tissue have been successfully used as positive controls in immunohistochemistry . For Western blot applications, whole-cell extracts from various cancer cell lines, particularly multiple myeloma cells and lung adenocarcinoma cells, have shown detectable ELOVL6 expression .

Appropriate antibody dilutions for Western blot range from 1 μg/mL to 2 μg/mL based on published protocols . When performing immunoblotting, ELOVL6 antibody (ABS458; EMD Millipore) has been used at 1:300 dilution in published research .

How should researchers troubleshoot specificity issues with ELOVL6 antibodies?

To address specificity concerns when working with ELOVL6 antibodies:

• Compare results with genetic knockdown models: Use ELOVL6 knockout or knockdown cells as negative controls. Research has shown that creating ELOVL6 knockout cells results in complete ablation of the protein, making them excellent negative controls for antibody validation .

• Cross-validation with multiple antibodies: Compare results using antibodies from different sources or targeting different epitopes of ELOVL6.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to verify signal specificity. For example, antibodies raised against synthetic peptides within human ELOVL6 should show reduced signal when pre-incubated with the immunogen .

• Test across species: Verify cross-reactivity claims by testing in samples from different species, noting that many ELOVL6 antibodies have been validated in human and rat samples but may work in other species based on sequence homology .

How does ELOVL6 expression correlate with cancer progression and patient prognosis?

Multiple studies have investigated ELOVL6's role in cancer progression and patient outcomes, revealing significant correlations:

In multiple myeloma (MM), lower levels of ELOVL6 have been observed in cells from bortezomib (BTZ) non-responsive patients compared to BTZ-responsive patients . Restoration of ELOVL6 levels in BTZ-resistant MM cells resensitized them to BTZ treatment, primarily through upregulation of ELOVL6-dependent ceramide species . This finding suggests ELOVL6 serves as a clinically relevant regulator of MM cell resistance to BTZ treatment .

Recent in vivo validation studies have shown that ELOVL6 inhibition significantly reduces tumor growth and enhances response to cancer treatments such as Abraxane, further supporting its role in cancer progression .

What methodological approaches are recommended for studying ELOVL6's role in tumor immune microenvironment?

ELOVL6 has been linked to immune infiltration in tumors, particularly in lung adenocarcinoma. Researchers investigating this relationship should consider:

• Comprehensive database analysis: Utilizing multiple databases such as TIMER, TISIDB, and GEPIA2 to analyze correlations between ELOVL6 expression and immune cell infiltration .

• Immune cell profiling: Studies show ELOVL6 expression positively correlates with CD8+ T cells, CD4+ T cells, myeloid-derived suppressor cells, regulatory T cells, and neutrophils, while negatively correlating with B cells in LUAD .

• Gene marker analysis: Examining correlations between ELOVL6 expression and immune marker genes, particularly those of regulatory T cells and myeloid-derived suppressor cells (positive correlation) and B cell marker genes (negative correlation) .

• Patient stratification: Grouping patients according to median ELOVL6 expression values to generate immune cell histograms and compare immunological profiles between high and low expression groups .

• Validation through immunohistochemistry: Using multiplexed IHC to simultaneously detect ELOVL6 and immune cell markers in tissue samples to confirm computational findings.

How can researchers effectively validate ELOVL6 inhibition or silencing at the molecular level?

When studying ELOVL6 inhibition or knockdown, several validation approaches have been validated in the literature:

• Genetic interference methods:

  • shRNA-mediated knockdown followed by Western blot validation of protein expression reduction

  • CRISPR/Cas9-mediated knockout resulting in complete ablation of ELOVL6 protein

• Pharmacological inhibition:

  • Treatment with specific inhibitors such as ELOVL6-IN-2, which has been shown to specifically target ELOVL6 activity

  • Comparison of inhibitor effects between wild-type and ELOVL6 knockout cells to confirm specificity

• Functional validation:

  • Cell proliferation assays showing reduced proliferation across tested cell lines following ELOVL6 interference

  • Colony formation assays demonstrating decreased colony growth

  • Cell cycle analysis revealing G1 phase accumulation without changes in apoptotic cell numbers

• Transcriptomic validation:

  • RNA-seq analysis to identify differentially expressed genes (202 identified in one study) following ELOVL6 inhibition

  • Gene Set Enrichment Analysis (GSEA) showing reduced enrichment in "myc targets" and "cell cycle" pathways in ELOVL6-inhibited cells

What are the key considerations when analyzing ELOVL6's impact on cellular lipid metabolism?

When investigating ELOVL6's role in lipid metabolism, researchers should consider:

• Fatty acid profiling: ELOVL6 primarily elongates fatty acids with 12, 14, and 16 carbons, showing higher activity toward C16:0 acyl-CoAs and catalyzing the synthesis of unsaturated C16 long-chain fatty acids . Mass spectrometry-based lipidomics approaches can quantify changes in fatty acid chain length distributions.

• Ceramide species analysis: ELOVL6 has been linked to specific ceramide species production, which is crucial for endoplasmic reticulum stress responses and cell death pathways, particularly in multiple myeloma cells .

• Membrane composition studies: ELOVL6 participates in producing VLCFAs that serve as precursors of membrane lipids, suggesting analysis of membrane composition changes following ELOVL6 manipulation .

• Metabolic pathway interactions: Consider ELOVL6's relationship with other lipid metabolism enzymes and transcription factors like SREBP1, which was included in immunoblotting panels alongside ELOVL6 in published studies .

• Cell-specific effects: Different cell types may show varied responses to ELOVL6 manipulation. For example, PDAC cells showed consistent reduction in proliferation following ELOVL6 interference, while effects on macropinocytosis and micropinocytosis were also observed .

How does ELOVL6 inhibition affect cellular processes beyond lipid metabolism?

Research has revealed that ELOVL6 inhibition impacts multiple cellular processes:

• Cell cycle regulation: ELOVL6 silencing or inhibition results in G1 phase cell cycle arrest without inducing apoptosis . This finding was confirmed at both the cellular and transcriptomic levels.

• Transcriptional programs: RNA-seq analysis identified 202 differentially expressed genes following ELOVL6 inhibition, with 142 showing downregulation . Gene Set Enrichment Analysis revealed reduced enrichment in "myc targets" and "cell cycle" pathways.

• Endocytic processes: ELOVL6 interference, either through shRNAs or chemical inhibition, resulted in increased micropinocytosis and macropinocytosis, as demonstrated by dextran-rhodamine B uptake assays .

• Drug sensitivity: ELOVL6 levels correlate with sensitivity to various drugs. In multiple myeloma, restoration of ELOVL6 levels resensitized resistant cells to bortezomib . In other cancer types, ELOVL6 inhibition enhanced response to treatments like Abraxane .

• Endoplasmic reticulum stress: ELOVL6-dependent ceramide species production appears to be a prerequisite for bortezomib-induced ER stress and cell death in multiple myeloma cells .

What experimental designs are most effective for studying ELOVL6's role in drug resistance?

Based on published research, effective experimental designs for studying ELOVL6's role in drug resistance include:

• Clinical sample comparison: Analyzing ELOVL6 expression in patient samples that responded differently to treatment. For example, comparing MM cells from bortezomib-responsive versus non-responsive patients revealed lower ELOVL6 levels in non-responsive cases .

• Gain and loss of function models:

  • Restoring ELOVL6 expression in resistant cell lines to determine if drug sensitivity is recovered

  • Knocking down ELOVL6 in sensitive cell lines to test if resistance develops

• Mechanism exploration:

  • Analyzing ceramide composition changes in response to drug treatment with and without ELOVL6 manipulation

  • Assessing ER stress markers (BiP, ATF4, XBP1s) in relation to ELOVL6 expression

• In vivo validation models:

  • Testing combination therapies of ELOVL6 inhibitors with standard cancer drugs like Abraxane

  • Monitoring tumor growth and treatment response in xenograft models with variable ELOVL6 expression

What are the recommended approaches for protein-protein interaction studies involving ELOVL6?

When investigating ELOVL6's protein interactions, researchers have employed several effective methodologies:

• Bioinformatic prediction: The STRING database has been used to construct protein interaction networks for ELOVL6 and its co-expressed genes .

• Network visualization: Cytoscape software (version 3.7.2) has been employed to visualize protein interaction networks, with color-coding to indicate up-regulated genes, down-regulated genes, and ELOVL6 .

• Co-expression analysis: Identifying the most significantly co-expressed genes with ELOVL6 provides insights into potential functional relationships and interaction partners .

• Experimental validation approaches should include:

  • Co-immunoprecipitation with ELOVL6 antibodies followed by mass spectrometry

  • Proximity labeling methods such as BioID or APEX to identify proteins in close proximity to ELOVL6 in its native cellular environment

  • Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) for detecting direct interactions with suspected partners

Of note, ELOVL6 is an endoplasmic reticulum-bound enzyme, which requires careful consideration of membrane protein interaction techniques and appropriate detergent conditions for solubilization.

What are the common technical challenges when detecting ELOVL6 in different sample types?

Researchers working with ELOVL6 detection should be aware of several technical considerations:

• Antibody specificity: Given ELOVL6's membership in the ELOVL family, cross-reactivity with other family members is a potential issue. Using antibodies validated against synthetic peptides specific to ELOVL6 can help ensure specificity .

• Tissue-specific expression: ELOVL6 expression varies across tissues, with notable detection in human hepatocirrhosis tissue and brain tissue . Antibody working dilutions may need adjustment based on the tissue being examined.

• Antigen retrieval methods: For IHC applications, different antigen retrieval buffers significantly impact detection quality. TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 provides an alternative .

• Detection in membrane fractions: As an endoplasmic reticulum-bound enzyme, proper sample preparation to retain membrane proteins is crucial for successful detection of ELOVL6 .

• Western blot optimization: When performing immunoblotting, ELOVL6 antibody concentrations between 1-2 μg/mL have been successfully employed , though specific antibodies may require different dilutions (e.g., ABS458 at 1:300 dilution) .

How can researchers differentiate between changes in ELOVL6 expression versus activity?

Distinguishing between ELOVL6 expression and enzymatic activity is critical for comprehensive functional studies:

• Expression assessment:

  • Western blot with ELOVL6 antibodies quantifies protein expression levels

  • qRT-PCR measures mRNA expression

  • Immunohistochemistry/immunofluorescence evaluates tissue or cellular distribution patterns

• Activity measurement approaches:

  • Metabolic labeling with radioactive or stable isotope-labeled fatty acid precursors

  • Mass spectrometry-based lipidomics to quantify substrate-to-product ratios

  • Focused analysis of specific fatty acid elongation products (particularly C16 to C18 conversion)

  • In vitro elongase activity assays using microsomal fractions

• Combined approaches:

  • Correlation analysis between expression levels and metabolic products

  • Comparing wild-type cells with those expressing catalytically inactive ELOVL6 mutants

  • Pharmacological inhibition with specific ELOVL6 inhibitors like ELOVL6-IN-2 compared to genetic knockdown

What emerging applications exist for ELOVL6 antibodies in cancer research?

Several promising research directions are emerging for ELOVL6 antibodies in cancer research:

How can researchers integrate ELOVL6 studies with broader metabolic research?

Integrating ELOVL6 research into broader metabolic studies requires several strategic approaches:

• Multi-omics integration: Combining transcriptomics, proteomics, and lipidomics data to understand how ELOVL6 expression changes affect the broader metabolic landscape. RNA-seq analysis has already identified 202 differentially expressed genes following ELOVL6 inhibition .

• Pathway cross-talk exploration: Investigating interactions between ELOVL6-mediated lipid metabolism and other metabolic pathways, particularly those regulated by c-MYC and other oncogenic drivers .

• Therapeutic combination strategies: Exploring how ELOVL6 inhibition might sensitize cells to metabolic therapies targeting complementary pathways. For example, ELOVL6 inhibition enhances response to Abraxane .

• Microenvironmental metabolism: Studying how ELOVL6-mediated changes in cancer cell metabolism affect immune cells in the tumor microenvironment, given ELOVL6's correlation with immune infiltration patterns .

• Single-cell approaches: Applying single-cell technologies to understand heterogeneity in ELOVL6 expression and its metabolic consequences within tumors and between different cell populations.