EXL5 Antibody

What is ELOVL5 and why is it significant in research?

ELOVL5 belongs to the elongase family of proteins responsible for catalyzing the rate-limiting step in the elongation of very long-chain fatty acids. This membrane-bound enzyme is particularly important in polyunsaturated fatty acid (PUFA) biosynthesis, functioning primarily in the elongation of C18-C20 PUFAs.

ELOVL5 has significant research value for several reasons:

• It plays a critical role in maintaining cellular membrane integrity and function

• It contributes to the synthesis of essential fatty acids that cannot be produced de novo by humans

• Dysregulation of ELOVL5 has been implicated in metabolic disorders, neurological conditions, and certain cancers

• It represents a potential therapeutic target for conditions involving lipid metabolism abnormalities

Current antibody-based approaches allow researchers to detect, quantify, and localize this protein in various experimental contexts, making them valuable tools for elucidating its biological functions and pathological implications.

What types of ELOVL5 antibodies are available for research applications?

Several types of ELOVL5 antibodies are currently available for research applications, each with distinct advantages:

• Polyclonal antibodies: These are derived from multiple B cell lineages and recognize multiple epitopes on the ELOVL5 protein. The most common are rabbit polyclonal anti-ELOVL5 antibodies designed for high specificity research applications .

• Monoclonal antibodies: Though not specifically mentioned in the search results for ELOVL5, these recognize a single epitope and offer higher specificity and batch-to-batch consistency compared to polyclonal antibodies.

• Recombinant antibodies: Produced using recombinant DNA technology, these offer exceptional consistency between batches and can be engineered for specific research needs.

The choice between these formats depends on the experimental requirements, with considerations including specificity, sensitivity, application compatibility, and reproducibility needs. Many commercially available ELOVL5 antibodies undergo rigorous validation processes to ensure reliable performance across multiple applications.

What are the validated applications for ELOVL5 antibodies?

ELOVL5 antibodies have been validated for multiple research applications, offering researchers flexibility in experimental design:

• Immunohistochemistry (IHC): Used for detecting ELOVL5 in tissue sections, allowing researchers to study expression patterns across different tissues and in pathological conditions .

• Immunocytochemistry/Immunofluorescence (ICC-IF): Enables visualization of ELOVL5 in cultured cells, providing insights into subcellular localization and expression dynamics under various experimental conditions .

• Western Blotting (WB): Allows quantification of ELOVL5 protein levels in cell or tissue lysates, particularly useful for comparative studies across different experimental treatments or disease states .

• Enzyme-Linked Immunosorbent Assay (ELISA): Permits quantitative detection of ELOVL5 in complex biological samples, though may require optimization due to the membrane-associated nature of the protein.

Each application requires specific optimization protocols to ensure reliable and reproducible results, particularly regarding sample preparation and antibody concentration.

How should I validate an ELOVL5 antibody before use in critical experiments?

Comprehensive validation is essential before using an ELOVL5 antibody in pivotal experiments. A methodical approach includes:

• Specificity verification:

  • Western blot analysis to confirm detection of a protein at the expected molecular weight (~35 kDa for human ELOVL5)

  • Peptide competition assays where pre-incubation with the immunizing peptide should abolish specific binding

  • Cross-reactivity assessment with other ELOVL family members (particularly ELOVL2 and ELOVL6 which have functional overlap)

• Control experiments:

  • Positive controls using tissues/cells known to express ELOVL5 (liver, adrenal glands, and sebaceous glands typically show high expression)

  • Negative controls using tissues with minimal ELOVL5 expression or ELOVL5 knockout/knockdown models

  • Secondary antibody-only controls to assess non-specific binding

• Analytical validation:

  • Serial dilution studies to determine optimal antibody concentration and confirm binding specificity, similar to the approach described for other antibodies: "The analytical specificity was confirmed by serial dilution and inhibition studies. As shown in Figures 5A–D, the binding of these antibodies declined significantly in proportion to the dilution of the antibody"

  • Inhibition studies where introducing soluble ELOVL5 antigen should competitively reduce antibody binding to immobilized targets

• Multi-method confirmation:

  • Comparing antibody performance across multiple detection methods (e.g., WB, IHC, ICC)

  • Comparing results with orthogonal approaches (e.g., mRNA expression data, mass spectrometry)

This systematic validation approach ensures reliable antibody performance and facilitates accurate interpretation of experimental results.

What are the optimal conditions for Western blot detection of ELOVL5?

Western blot optimization for ELOVL5 detection requires attention to several technical considerations:

• Sample preparation:

  • Use membrane protein extraction buffers containing appropriate detergents (RIPA or NP-40 based buffers with 0.1-0.5% SDS)

  • Include protease inhibitors to prevent degradation

  • Consider membrane fraction enrichment techniques for improved sensitivity

  • Heat samples at 70°C (not 95°C) to prevent membrane protein aggregation

• Gel electrophoresis parameters:

  • Use 10-12% polyacrylamide gels for optimal resolution of ELOVL5 (~35 kDa)

  • Load 20-50 μg of total protein per lane for standard detection

  • Include molecular weight markers spanning 25-50 kDa range

• Transfer and blocking conditions:

  • Semi-dry or wet transfer systems (wet transfer often provides better results for membrane proteins)

  • PVDF membranes typically yield better results than nitrocellulose for membrane proteins

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody dilution: Start with manufacturer's recommendation (typically 1:500 to 1:2000)

  • Incubate overnight at 4°C with gentle agitation

  • Secondary antibody dilution: Typically 1:5000 to 1:10000

  • Consider signal amplification systems for low-abundance detection

• Detection and quantification:

  • Use enhanced chemiluminescence (ECL) or near-infrared fluorescence detection

  • Include appropriate loading controls (e.g., Na⁺/K⁺-ATPase or calnexin for membrane fractions)

  • Perform densitometric analysis with normalization to loading controls

Optimization should include a dilution series test similar to that described in the literature: "Anti-spike protein antibody reacting with spike protein at a dilution of 1:200 gave an OD of 3.4, a dilution of 1:800 gave an OD of 2.6, and a dilution of 1:25600 resulted in an OD of 0.39" . The same principle applies to optimizing ELOVL5 antibody dilutions.

How can I optimize immunohistochemistry protocols for ELOVL5 detection in tissue samples?

Optimizing immunohistochemistry for ELOVL5 requires methodical protocol development:

• Tissue preparation and fixation:

  • 10% neutral buffered formalin fixation (18-24 hours) typically preserves ELOVL5 antigenicity

  • Paraffin embedding followed by 4-6 μm section thickness

  • Use positively charged slides to prevent tissue detachment

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Test both pressure cooker (2-3 minutes) and microwave methods (15-20 minutes)

  • Allow slides to cool slowly to room temperature after retrieval

• Blocking and antibody conditions:

  • Block endogenous peroxidase activity with 3% H₂O₂ (10 minutes)

  • Block non-specific binding with 5-10% normal serum from secondary antibody species

  • Primary antibody dilution: Begin with 1:100-1:500 dilution

  • Incubation conditions: 1 hour at room temperature or overnight at 4°C

  • Secondary antibody systems: HRP-polymer detection systems typically provide better signal-to-noise ratio

• Signal development and counterstaining:

  • DAB development: Monitor under microscope to determine optimal time (typically 1-5 minutes)

  • Hematoxylin counterstaining: Light counterstaining to avoid obscuring ELOVL5 signal

  • Aqueous mounting for initial evaluation, permanent mounting for long-term storage

• Controls and validation:

  • Include positive control tissues with known ELOVL5 expression (liver sections are recommended)

  • Include negative controls (primary antibody omission and isotype controls)

  • Validate staining pattern against published literature

• Quantification approach:

  • Define scoring criteria (intensity, percentage positive cells, H-score)

  • Use digital image analysis software for objective quantification

  • Have multiple trained observers score independently

This systematic optimization approach ensures reliable detection of ELOVL5 in tissue samples while minimizing background and non-specific staining.

How do I quantify ELOVL5 expression levels in experimental samples?

Accurate quantification of ELOVL5 requires appropriate methodological approaches depending on the technique:

• Western blot quantification:

  • Use digital densitometry software (ImageJ, Image Studio, etc.)

  • Include a standard curve using recombinant ELOVL5 protein if absolute quantification is required

  • Normalize to appropriate loading controls (Na⁺/K⁺-ATPase for membrane proteins)

  • Include at least three biological replicates for statistical analysis

  • Apply appropriate statistical tests (t-test for two groups, ANOVA for multiple groups)

• Immunohistochemistry quantification:

  • Use digital pathology software for quantitative analysis

  • Quantify using parameters such as:

    • H-score = Σ (percentage of cells with intensity i) × (i), where i = 0, 1, 2, 3

    • Percentage of positive cells

    • Mean optical density

  • Establish regions of interest (ROIs) consistently across samples

  • Validate computer-assisted quantification against manual scoring by pathologists

• ELISA-based quantification:

  • Generate standard curves using purified ELOVL5 protein

  • Ensure parallel dilution behavior between standards and samples

  • Calculate concentration using the formula described in similar antibody research contexts:
    "The percentage of tissue reaction with each antibody was calculated based on the following formula..."

  • Include spike-recovery experiments to validate quantification in complex matrices

• Flow cytometry quantification:

  • Use mean fluorescence intensity (MFI) for relative quantification

  • Calculate fold change compared to control samples

  • Consider using calibration beads for absolute quantification

Regardless of method, statistical validation through appropriate replicates and statistical tests is essential for reliable quantification.

What controls are essential for ensuring reliable ELOVL5 antibody-based experiments?

A comprehensive control strategy is critical for generating reliable data with ELOVL5 antibodies:

• Experimental controls:

  • Positive tissue/cell controls: Samples known to express ELOVL5 (liver, sebaceous glands)

  • Negative tissue/cell controls: Samples with minimal ELOVL5 expression

  • Genetic controls: ELOVL5 knockout/knockdown models where available

  • Overexpression controls: Cells transfected with ELOVL5 expression vectors

• Technical controls:

  • Primary antibody omission: To assess non-specific binding of secondary antibody

  • Isotype controls: Non-specific IgG from the same species as primary antibody

  • Secondary antibody-only controls: To identify non-specific binding

  • Loading controls: For normalization in Western blot (Na⁺/K⁺-ATPase for membrane proteins)

• Specificity controls:

  • Peptide competition/blocking: Pre-incubation of antibody with immunizing peptide

  • Multiple antibody validation: Use of antibodies targeting different ELOVL5 epitopes

  • Cross-reactivity assessment: Testing against other ELOVL family members

• Quantification controls:

  • Standard curves: Using recombinant ELOVL5 protein

  • Internal reference standards: Consistent positive control samples across experiments

  • Inter-assay calibrators: To normalize between experimental batches

This approach follows established validation principles: "Furthermore, the antibodies and other reagents were added to four wells coated with 2% HSA and four wells coated with 2% BSA alone; these were then used as negative controls. After the addition of other reagents to these control wells, the ODs were measured" . Similar control strategies should be employed for ELOVL5 antibody experiments.

How do I address cross-reactivity concerns with ELOVL5 antibodies?

Cross-reactivity with other ELOVL family members or unrelated proteins is a significant challenge that requires systematic investigation:

• Identifying potential cross-reactivity:

  • Sequence homology analysis: ELOVL5 shares significant homology with ELOVL2 (~60%) and ELOVL6 (~45%)

  • Expression pattern analysis: Compare observed patterns with known ELOVL5 expression profiles

  • Unexpected band detection: Multiple bands in Western blot may indicate cross-reactivity

  • Knockout/knockdown validation: Persistent signal in ELOVL5-depleted samples suggests cross-reactivity

• Experimental approaches to assess cross-reactivity:

  • Comparative testing: Evaluate antibody against recombinant proteins of all ELOVL family members

  • Inhibition studies: "To further demonstrate the specificity of these antibody reactions, an inhibition study was performed by the addition of M2, MBP, NFP, and GAD-65 in concentrations ranging from 0 to 128 micrograms into the liquid phase of the ELISA plates that were coated with the same antigen" - similar approaches can be applied for ELOVL5

  • Serial dilution testing: "The binding of these antibodies to 4 different SARS-CoV-2 proteins and cross-reactive antigens declined significantly in proportion to the dilution of the antibody" - apply this principle to ELOVL5 antibody validation

• Strategies to minimize cross-reactivity effects:

  • Epitope selection: Choose antibodies targeting unique regions of ELOVL5

  • Pre-adsorption: Pre-incubate antibodies with potential cross-reactive proteins

  • Increased stringency: Higher dilutions of primary antibody and more stringent washing

  • Alternative detection: Use multiple antibodies targeting different epitopes

• Data interpretation accounting for potential cross-reactivity:

  • Multi-method validation: Confirm key findings using orthogonal methods (mRNA analysis, mass spectrometry)

  • Genetic models: Use ELOVL5 knockout/knockdown models to confirm specificity

  • Computational prediction: Utilize tools that predict antibody cross-reactivity based on epitope analysis

The decline in signal with serial dilution can be quantified as shown in published research: "For example, anti-spike protein antibody reacting with spike protein at a dilution of 1:200 gave an OD of 3.4, a dilution of 1:800 gave an OD of 2.6, and a dilution of 1:25600 resulted in an OD of 0.39" . Similar patterns should be observed with specific ELOVL5 antibodies.

How can I use ELOVL5 antibodies for co-localization studies with other proteins?

Co-localization studies require careful planning and technical considerations:

Species compatibility is critical for co-localization: "Species switching has grown in popularity for in vitro research due to its ability to increase compatibility with a secondary antibody, enable easier co-labeling studies and prevent unwanted antibody interactions in serological assays" . This principle is directly applicable to designing ELOVL5 co-localization experiments.

Can ELOVL5 antibodies be modified for specialized research applications?

Several modification strategies can enhance ELOVL5 antibody utility for specialized applications:

• Fragment generation:

  • Fab fragments: Smaller size for improved tissue penetration and reduced cross-linking

  • F(ab')2 fragments: Bivalent binding without Fc-mediated effects

  • scFv (single-chain variable fragments): Minimal binding domains for specialized applications

  • Each format offers distinct advantages for specific experimental contexts

• Conjugation options:

  • Fluorophore conjugation: Direct fluorescent labeling eliminates secondary antibody needs

    • Alexa Fluor dyes (488, 555, 647) for standard fluorescence microscopy

    • Near-infrared dyes (IRDye 800CW) for in vivo imaging applications

  • Enzyme conjugation: HRP or AP for enhanced sensitivity in IHC/Western blot

  • Biotin labeling: For versatile detection with streptavidin systems

  • Nanoparticle conjugation: For imaging or therapeutic research applications

• Antibody engineering approaches:

  • Isotype switching: "For example, an IgG antibody, the major antibody of the secondary immune response, can be reformatted to an IgM antibody, the predominant antibody of the primary immune response, to aid in infectious disease research and diagnostic assay development"

  • Species switching: "Species switching involves reformatting the variable regions to an antibody backbone of a different species"

  • Affinity maturation: Enhancing binding affinity through targeted mutations

  • Stability engineering: Improving thermal and pH stability for challenging conditions

• Advanced formats:

  • Bispecific antibodies: Targeting ELOVL5 alongside another protein of interest

  • "This standard design is known as a 1:1 binder but you can also generate 2:1 and 2:2 binders... For some targets, more binding arms may be better to increase avidity"

  • Recombinant fusion proteins: Combining ELOVL5-binding domains with functional moieties

Each modification strategy requires validation to ensure retained specificity and functionality after the modification process.

What computational approaches can enhance ELOVL5 antibody research?

Emerging computational methods offer powerful tools for enhancing ELOVL5 antibody research:

• Epitope prediction and antibody design:

  • In silico epitope mapping of ELOVL5 to identify optimal target regions

  • Computational antibody design targeting unique ELOVL5 epitopes

  • Structure-based optimization of antibody-antigen interactions

  • Prediction of post-translational modifications that might affect antibody binding

• Cross-reactivity prediction:

  • Sequence-based analysis of potential cross-reactive targets

  • Structural modeling of antibody-antigen complexes

  • Machine learning approaches to predict off-target binding

  • "We develop a computational framework that predicts how an antibody or serum would inhibit any variant from any other study" - similar approaches could predict ELOVL5 antibody specificity

• Data integration and interpretation:

  • Machine learning for pattern recognition in antibody-based experimental data

  • Network analysis of ELOVL5 interactions based on co-localization data

  • Pathway modeling incorporating ELOVL5 quantitative expression data

  • Multi-omics data integration for comprehensive biological context

• Experimental design optimization:

  • Computational optimization of antibody concentration and incubation conditions

  • Statistical power analysis for determining optimal sample sizes

  • Predictive modeling to estimate assay performance

  • "Our approach paves the way to rationally design virus panels in future studies, saving time and resources by measuring a substantially smaller set of viruses" - similar principles apply to optimizing ELOVL5 antibody experiments

The low-dimensional nature of antibody-antigen interactions facilitates computational approaches: "Previous work has shown that antibody-virus inhibition data are intrinsically low dimensional, which spurred applications ranging from antigenic maps to the recovery of missing values from partially observed data" . These principles can be adapted to ELOVL5 antibody research for enhanced experimental design and data interpretation.

How do I troubleshoot weak or absent ELOVL5 signal in Western blot experiments?

Systematic troubleshooting can resolve weak or absent ELOVL5 signals:

• Sample preparation issues:

  • Inefficient protein extraction: Enhance extraction with specialized membrane protein buffers containing 1-2% SDS or other ionic detergents

  • Protein degradation: Use fresh samples, include protease inhibitors, maintain samples at 4°C

  • Insufficient protein loading: Increase loading to 40-60 μg per lane

  • Inappropriate sample heating: Use 70°C for 10 minutes instead of 95°C to prevent membrane protein aggregation

• Transfer problems:

  • Inefficient transfer: Optimize transfer conditions (increase time/voltage for membrane proteins)

  • Protein loss: Use PVDF membranes (0.45 μm pore size) for better protein retention

  • Transfer verification: Use reversible protein stains (Ponceau S) to confirm transfer

• Antibody-related issues:

  • Insufficient antibody concentration: Increase primary antibody concentration (try 1:200-1:500)

  • Antibody degradation: Use fresh aliquots, avoid repeated freeze-thaw cycles

  • Insufficient incubation: Extend primary antibody incubation to overnight at 4°C

  • Secondary antibody mismatch: Verify compatibility between primary and secondary antibodies

• Detection limitations:

  • Insufficient sensitivity: Use enhanced chemiluminescence substrate or switch to more sensitive detection systems

  • Signal development time: Extend exposure time for weak signals

  • Detection system failure: Include positive controls for detection system functionality

This systematic approach follows established troubleshooting principles for antibody-based detection methods and addresses the specific challenges of membrane protein detection.

How can I implement quality control measures for longitudinal ELOVL5 antibody studies?

Longitudinal studies require robust quality control to ensure consistency:

• Antibody quality control:

  • Maintain antibody aliquots at -20°C or -80°C to prevent degradation

  • Use single-use aliquots to avoid freeze-thaw cycles

  • Test new antibody lots against reference standards before implementation

  • Document lot numbers and maintain sample antibodies from each lot

• Standardization approaches:

  • Include consistent positive and negative controls across all experiments

  • Maintain reference standard samples (lysates/tissues with known ELOVL5 expression)

  • Use calibration standards for quantitative assays

  • Standardize all protocol steps with detailed SOPs

• Technical validation measures:

  • Implement replicate testing (technical and biological)

  • Calculate coefficients of variation to monitor assay performance

  • Use statistical process control charts to track assay performance over time

  • Implement regular proficiency testing if multiple operators are involved

• Data integration and traceability:

  • Maintain comprehensive documentation of experimental conditions

  • Use laboratory information management systems (LIMS) for data tracking

  • Implement unique identifiers for all samples and experiments

  • Establish criteria for data acceptance/rejection

This systematic approach ensures consistency in longitudinal studies, a critical factor for reliable ELOVL5 research spanning extended timeframes.

How might single-cell approaches enhance ELOVL5 antibody research?

Single-cell technologies offer unprecedented insights into cellular heterogeneity:

• Single-cell protein analysis technologies:

  • Mass cytometry (CyTOF): Metal-conjugated ELOVL5 antibodies for high-parameter analysis

  • Single-cell Western blotting: For quantifying ELOVL5 in individual cells

  • Imaging mass cytometry: For spatial analysis of ELOVL5 at single-cell resolution

  • Proximity extension assays: For sensitive detection in limited material

• Multimodal single-cell analysis:

  • Combined protein-mRNA analysis (CITE-seq): Correlating ELOVL5 protein with transcriptome

  • Spatial transcriptomics with protein detection: Mapping ELOVL5 in tissue spatial context

  • Single-cell proteomics with antibody-based enrichment: For deeper proteomic profiling

  • Metabolic-protein correlations: Linking ELOVL5 expression to cellular lipid profiles

• Computational integration approaches:

  • Machine learning algorithms to identify ELOVL5-expressing cell populations

  • Trajectory analysis to map ELOVL5 expression during cellular differentiation

  • Network analysis to identify ELOVL5-associated protein interactions

  • "Using interpretable machine learning to extend heterogeneous..." approaches for single-cell data integration

• Technical considerations:

  • Antibody validation at single-cell level (specificity in dilute conditions)

  • Sample preparation optimization for membrane protein preservation

  • Computational correction for technical artifacts

  • Data normalization approaches for quantitative comparison

These approaches could transform understanding of ELOVL5 biology by revealing cell-type-specific expression patterns and functional heterogeneity previously masked in bulk analyses.

What are the latest technologies for improving ELOVL5 antibody specificity and sensitivity?

Emerging technologies are enhancing antibody performance metrics:

• Advanced antibody engineering approaches:

  • Structure-guided antibody design targeting unique ELOVL5 epitopes

  • Deep mutational scanning to identify optimal binding variants

  • Computational affinity maturation for enhanced sensitivity

  • Novel scaffold platforms beyond traditional antibody formats

• Next-generation recombinant technologies:

  • High-throughput screening of antibody libraries against ELOVL5

  • Synthetic antibody libraries with optimized frameworks

  • Single B-cell cloning from immunized animals for native paired sequences

  • Transgenic animals expressing diverse human antibody repertoires

• Enhanced validation technologies:

  • CRISPR-based knockout validation in relevant cell types

  • Orthogonal target verification using mass spectrometry

  • Multiplexed epitope mapping using peptide arrays or hydrogen-deuterium exchange

  • Native protein interaction measurements using advanced biophysical techniques

• Manufacturing improvements:

  • Consistent recombinant production systems

  • "Manufacturability of an antibody is an important component to consider early on in the project. In some instances, developability concerns that are identified early can potentially be engineered out during the early-stage research phase"

  • Enhanced purification strategies for improved homogeneity

  • Stability engineering for extended shelf-life

These technologies collectively address the fundamental challenges of specificity and sensitivity in ELOVL5 antibody research, enabling more reliable and reproducible experimental outcomes.