ELOVL4 Antibody

What is the optimal dilution range for ELOVL4 antibody applications in neural tissue research?

ELOVL4 antibody dilution varies depending on the specific application and tissue type. For Western blotting, a dilution range of 1:500-1:1000 is typically effective for detecting the protein's 37kDa band in neural tissues. For immunohistochemistry on paraffin-embedded brain sections, a dilution range of 1:250-1:1000 works well, particularly when using TE buffer (pH 9.0) for antigen retrieval. Immunofluorescence applications typically require more concentrated antibody solutions, with effective ranges between 1:10-1:100 for cultured cells and 1:300-1:500 for brain tissue sections .

When optimizing dilutions, consider these tissue-specific observations:

• Mouse brain tissue typically shows strong signals at 1:500 dilution in Western blots

• Human brain samples may require slightly more concentrated solutions (1:300-1:400)

• For immunofluorescence in neural tissue, methanol fixation at -30°C for 20 minutes prior to antibody application significantly improves ELOVL4 labeling

Which brain regions show the strongest ELOVL4 expression when using immunohistochemistry?

Immunohistochemical mapping reveals region-specific ELOVL4 expression throughout the mouse brain. The highest expression levels are observed in:

• Cerebral cortex - particularly in neuronal cell bodies rather than neuropil

• Hippocampus - with prominent labeling in pyramidal neurons

• Thalamus and hypothalamus - showing variable expression across subnuclei

• Cerebellum - with strong expression in Purkinje cells

• Midbrain structures - including superior and inferior colliculi

Notable differences in expression intensity include:

• Strong labeling in the lateral preoptic area and lateral mammillary nucleus

• Prominent labeling in midbrain reticular nucleus

• Reduced expression in substantia nigra compared to surrounding midbrain regions

• Minimal expression in basal ganglia of the postnatal brain (in contrast to high expression during embryonic development)

The expression pattern is primarily neuronal, with less labeling in white matter tracts and minimal labeling in astrocytes or radial glial cells .

What are the recommended sample preparation protocols for optimal ELOVL4 antibody performance?

Effective sample preparation significantly impacts ELOVL4 antibody performance across different experimental applications:

For Western Blotting:

• Protein extraction from tissues should use RIPA buffer with protease inhibitors

• For neural tissues, homogenization at 4°C followed by centrifugation at 14,000g for 15 minutes yields optimal results

• Protein samples should be denatured at 95°C for 5 minutes in sample buffer prior to loading

• Blocking with 5% non-fat dry milk in PBST is recommended

• Primary antibody incubation should be performed overnight at 4°C

For Immunohistochemistry/Immunofluorescence:

• For frozen brain sections, immersion in 100% methanol at -30°C for 20 minutes significantly improves ELOVL4 labeling

• For paraffin-embedded tissues, antigen retrieval in 10mM citrate buffer (pH 6.0) heated to 95°C for 30-60 minutes is effective

• Alternative antigen retrieval using TE buffer (pH 9.0) has shown superior results in some tissues

• Blocking solution containing 2-10% normal goat serum, 5% bovine serum albumin, 1% fish gelatin, and 0.1-0.5% Triton X-100 in HBSS is effective

• Primary antibody incubation overnight at room temperature yields optimal results

How can I differentiate between wild-type and mutant ELOVL4 using antibodies in disease models?

Distinguishing between wild-type and mutant ELOVL4 proteins requires careful consideration of antibody epitope location and protein characteristics:

Antibody Selection Strategy:

• Choose antibodies targeting epitopes N-terminal to mutation sites, as STGD3-related mutations typically occur in exon 6

• The truncated mutant ELOVL4 protein (~33kDa) can be differentiated from wild-type (37kDa) using Western blot analysis

• Antibodies raised against the C-terminal region will not detect truncated mutant proteins

Experimental Approach:

• For Western blotting: Use gradient gels (10-12% polyacrylamide) to achieve better separation of wild-type and mutant proteins

• For heterozygous models: Both wild-type (37kDa) and mutant (33kDa) bands should be detectable on the same blot when using N-terminal targeting antibodies

• For immunofluorescence: Wild-type ELOVL4 localizes to the ER, while mutant proteins show punctate aggregation patterns due to loss of ER retention signals

Validation and Controls:

• Include homozygous wild-type, heterozygous, and homozygous mutant samples when possible

• In cellular models, co-expression experiments with tagged wild-type and mutant constructs can confirm dominant-negative effects

• Immunoprecipitation followed by Western blotting can confirm protein-protein interactions between wild-type and mutant forms

What are the considerations for dual-labeling experiments involving ELOVL4 and other cellular markers?

When designing dual-labeling experiments to study ELOVL4 in relation to other cellular components, several technical considerations are crucial:

Antibody Compatibility:

• Primary antibodies must be raised in different host species (e.g., rabbit anti-ELOVL4 with mouse anti-cellular marker)

• If using same-species antibodies, sequential immunolabeling with complete blocking steps is required

Cellular Markers Successfully Co-labeled with ELOVL4:

Protocol Optimization:

• Use methanol fixation (-30°C for 20 minutes) for ELOVL4 labeling

• Confirm compatibility of fixation method with other markers

• For sequential labeling, complete the ELOVL4 labeling first

• Use highly cross-adsorbed secondary antibodies to prevent cross-reactivity

• Include appropriate controls: single labeling controls and secondary-only controls

How does ELOVL4 expression change during neural development, and what are the best methods to study these changes?

ELOVL4 shows distinct spatiotemporal expression patterns during neural development, requiring stage-specific optimization of antibody techniques:

Developmental Expression Pattern:

• ELOVL4 is widely expressed in the developing brain by embryonic day 18

• Intense expression occurs in regions underlying lateral ventricles and other neurogenic regions

• The basal ganglia shows particularly strong expression in embryonic stages but minimal expression postnatally

• Peak mRNA expression occurs around postnatal day 1 (P1), declining by P30 before reaching steady-state levels

Methodological Approaches:

• For Embryonic Tissues:

  • Cryosections of unfixed tissue with methanol post-fixation (-30°C) yield superior results

  • More concentrated antibody dilutions (1:200-1:300) may be required for embryonic tissue

• For Postnatal Development Studies:

  • Comparative analysis across multiple timepoints (e.g., P4, P14, P60) with consistent protocols

  • Co-labeling with developmental markers (proliferation markers, maturation markers)

  • Quantitative approaches to measure expression changes:

    • Western blot analysis with age-matched loading controls

    • Quantitative immunofluorescence with standardized image acquisition parameters

• Technical Considerations:

  • Tissue fixation must be carefully controlled across developmental stages

  • Background autofluorescence increases with age in many neural tissues

  • Region-specific analysis is crucial as developmental trajectories vary across brain regions

What controls are essential when validating the specificity of ELOVL4 antibodies?

Rigorous validation of ELOVL4 antibodies requires multiple complementary approaches:

Essential Controls:

• Genetic Controls:

  • Tissue from ELOVL4 knockout or knockdown models (conditional knockouts are available)

  • Comparative analysis of tissues with known differential expression (high: retina, skin; low: heart, liver)

  • Heterologous expression systems (cells transfected with ELOVL4 vs. control vectors)

• Biochemical Controls:

  • Peptide blocking experiments - pre-incubation of antibody with immunizing peptide should abolish signal

  • Western blot showing single band at expected molecular weight (37kDa for wild-type ELOVL4)

  • Sequential dilution series to demonstrate signal specificity and sensitivity

• Methodological Controls:

  • Omission of primary antibody while maintaining all other steps

  • Substitution with non-specific IgG from the same host species

  • Comparison of multiple antibodies targeting different epitopes of ELOVL4

Validation Data Example:
When validating antibody specificity in neural tissues, researchers observed:

• A single 37kDa band in wild-type mouse retina that was absent in conditional knockout models

• Specific labeling in photoreceptor inner segments that was abolished by peptide blocking

• Overlapping patterns between immunohistochemistry and in situ hybridization data

What are the most common technical challenges when working with ELOVL4 antibodies and how can they be overcome?

Researchers frequently encounter several challenges when working with ELOVL4 antibodies that can be addressed through specific protocol modifications:

Challenge 1: Weak or Absent Signal in Immunostaining

• Solution: Enhanced antigen retrieval is critical. For brain tissue, methanol treatment at -30°C for 20 minutes significantly improves ELOVL4 labeling. For paraffin sections, try TE buffer (pH 9.0) as an alternative to citrate buffer.

• Approach: Increase antibody concentration and extend incubation time to overnight at room temperature rather than 4°C

Challenge 2: High Background in Western Blots

• Solution: Optimize blocking conditions using 5% non-fat dry milk in PBST. Extend blocking time to overnight at 4°C.

• Approach: Include additional washing steps (4 times, 10 minutes each) and use higher dilutions of secondary antibody (1:10,000 to 1:20,000)

Challenge 3: Inconsistent Results Across Different Tissue Types

• Solution: Tissue-specific optimization is necessary. The optimal antibody dilution for retina (1:300-1:500) may differ from that for brain (1:500-1:1000).

• Approach: Perform dilution series for each new tissue type and consider tissue-specific fixation protocols

Challenge 4: Difficulty Detecting ELOVL4 in Certain Brain Regions

• Solution: Some brain regions (e.g., basal ganglia in adult brain) naturally express very low levels of ELOVL4. Increase sensitivity using amplification systems such as tyramide signal amplification.

• Approach: Extend primary antibody incubation times and use more sensitive detection systems

How can I design experiments to study the relationship between ELOVL4 expression and its enzymatic products (VLC-FAs and VLC-PUFAs)?

Investigating the relationship between ELOVL4 protein expression and its enzymatic products requires an integrated experimental approach:

Experimental Design Strategy:

• Parallel Analysis of Protein and Lipids:

  • Divide tissue samples for simultaneous protein extraction (for ELOVL4 immunoblotting) and lipid extraction (for fatty acid analysis)

  • For cellular models, perform immunocytochemistry in sister cultures used for lipid analysis

• Fatty Acid Analysis Methods Compatible with Immunological Studies:

  • GC-MS analysis of fatty acid methyl esters (FAMEs) for VLC-FA detection

  • HPLC-MS for analysis of complex lipids containing VLC-PUFAs

  • Tissues require specialized extraction methods to preserve both protein integrity and lipid profiles

• Correlation Approaches:

  • Quantitative Western blotting for ELOVL4 protein levels

  • Quantitative mass spectrometry for VLC-FA and VLC-PUFA levels

  • Statistical correlation analysis between protein expression and fatty acid levels

  • Region-specific analysis in brain to correlate expression patterns with lipid composition

Model Systems and Manipulations:

• Gain-of-Function: Adenoviral transduction of cells with ELOVL4 shows direct evidence of enzymatic activity

  • Cardiomyocytes and ARPE-19 cells transduced with ELOVL4 produce C28-C30 saturated fatty acids

  • Supplementation with precursors (24:0, 20:5n3, or 22:5n3) demonstrates specific elongation products

• Loss-of-Function: Conditional knockout models show:

  • Specific decreases in VLC-PUFAs in retinal phospholipids

  • Particular reduction in phosphatidylcholine species containing VLC-PUFAs at the sn-1 position

  • Accompanying functional deficits that correlate with lipid changes

How do I interpret differences in ELOVL4 expression between immunohistochemistry and Western blot results?

Discrepancies between immunohistochemistry and Western blot results for ELOVL4 require careful consideration of several factors:

Common Discrepancy Scenarios and Interpretations:

Resolution Strategies:

• Technical Verification:

  • Confirm antibody specificity using knockout/knockdown controls in both applications

  • Verify that sample preparation preserves protein integrity (add protease inhibitors)

  • Ensure antigen retrieval methods are optimized for ELOVL4 detection

• Biological Interpretation:

  • Consider subcellular localization - ELOVL4 is primarily localized to the ER

  • Region-specific expression variation may affect whole-tissue Western blot results

  • Developmental stage differences may explain apparent discrepancies

What factors should be considered when quantifying ELOVL4 expression across different brain regions?

Accurate quantification of ELOVL4 expression across brain regions requires consideration of multiple variables:

Methodological Considerations:

Quantification Approaches:

• For Western Blotting:

  • Precise dissection of anatomical regions

  • Normalization to appropriate loading controls (β-actin often varies between regions)

  • Consider specialized normalization to neuron-specific markers for primarily neuronal proteins

• For Immunofluorescence:

  • Cell counting approaches (% positive cells)

  • Mean fluorescence intensity measurements

  • Colocalization analysis with cell-type markers

  • 3D reconstruction for volumetric analysis

Biological Variables to Consider:

• Significant expression differences exist between brain regions (e.g., strong in cortex, weak in basal ganglia)

• Cell-type composition varies dramatically across regions

• Developmental stage dramatically affects expression patterns

• ELOVL4 is primarily expressed in neurons, with limited expression in oligodendrocytes and minimal expression in astrocytes

How can I determine if observed changes in ELOVL4 immunolabeling are due to changes in protein expression versus alterations in subcellular localization?

Distinguishing between changes in expression level and altered subcellular distribution requires specialized approaches:

Experimental Design for Differentiation:

• Combined Approaches:

  • Pair Western blot analysis (for total protein levels) with immunolocalization studies

  • Subcellular fractionation followed by Western blotting of different cellular compartments

  • High-resolution microscopy with quantitative spatial analysis

• Subcellular Markers for Co-localization:

  • ER markers (calnexin, PDI) - normal ELOVL4 localization

  • Golgi markers (GM130)

  • Aggresome markers (ubiquitin, p62) - for mislocalized/aggregated protein

  • Quantitative colocalization analysis with appropriate statistical methods

Analytical Framework:

Specific Case of ELOVL4 Mutations:

• Wild-type ELOVL4 localizes to the ER due to its C-terminal dilysine motif

• Mutant ELOVL4 missing this motif shows punctate, non-ER distribution

• When co-expressed, mutant protein can cause wild-type to mislocalize through protein-protein interactions

• This dominant-negative effect can be monitored through quantitative colocalization studies