ACSL6 Antibody, HRP conjugated

What is ACSL6 and what cellular functions does it perform?

ACSL6 is an enzyme that catalyzes the formation of acyl-CoA from fatty acids, ATP, and CoA, utilizing magnesium as a cofactor. This reaction is critical for activating fatty acids for subsequent metabolic processes. ACSL6 plays a major role in fatty acid metabolism, particularly in the brain, where it contributes to lipid homeostasis . The enzyme preferentially processes specific fatty acid substrates, including palmitoleate, oleate, and linoleate . In certain tissues, ACSL6 demonstrates substrate preferences between arachidonate and other eicosanoid derivatives such as epoxyeicosatrienoic acids (EETs) or hydroxyeicosatrienoic acids (HETEs) .

The biological significance of ACSL6 extends beyond basic metabolism. Recent research has demonstrated that ACSL6 mRNA expression in skeletal muscle is modulated by nutritional status, with exercise and fasting decreasing ACSL6 mRNA levels, while acute lipid ingestion increases its expression . Furthermore, studies have shown that ACSL6 gene inhibition in rat primary myotubes decreases lipid accumulation and activates mitochondrial oxidative capacity through the AMPK/PGC1-α pathway .

What are the different variants of ACSL6 and how do they differ functionally?

ACSL6 exists in multiple isoforms resulting from alternative splicing events. Research has identified several transcript variants with distinct functional properties:

These variants show differential expression patterns across tissues, suggesting tissue-specific functions. For instance, when analyzing protein extracts from the erythroleukemic cell line K562, ACSL6 antibody recognized three major species: one at 75 kDa and two at 130 kDa . The molecular basis for this variation involves alternative splicing events that can remove the Gate domain and alter membrane association properties .

What is the significance of HRP conjugation for ACSL6 antibody applications?

Horseradish peroxidase (HRP) conjugation represents a critical modification for ACSL6 antibodies that enhances their utility in multiple research applications. The conjugation process creates a direct linkage between the antibody and the enzyme, eliminating the need for secondary antibody detection systems. This modification provides several methodological advantages:

• Enhanced sensitivity in Western blotting applications, allowing for detection of ACSL6 even when present at low concentrations

• Reduced background signal compared to two-step detection systems

• Simplified experimental workflows in immunodetection protocols

• Compatibility with various substrates for colorimetric, chemiluminescent, or fluorescent detection

For example, ACSL6 antibody with HRP conjugation (such as ARP41858_P050-HRP) is supplied at 0.5 mg/ml concentration and is particularly optimized for Western blotting applications . The direct conjugation to HRP enables sensitive detection of the target protein (68 kDa) across multiple species including human, mouse, and rat samples .

How can different detection methods be optimized when using ACSL6 antibody, HRP conjugated?

Optimizing detection protocols with HRP-conjugated ACSL6 antibodies requires careful consideration of several experimental variables:

For Western blotting applications:

• Sample preparation: Total protein extracts for ACSL6 detection benefit from specific handling protocols. Research indicates that the high molecular weight band (130 kDa) of ACSL6 may not be detected when protein samples are boiled before loading or when samples are overly diluted in SDS-PAGE loading buffer . To preserve these higher molecular weight species, protein denaturation should be performed at 37°C for 20 minutes rather than boiling .

• Membrane blocking: 5% non-fat dry milk in TBST has proven effective for reducing background signal during ACSL6 immunodetection .

• Antibody dilution: HRP-conjugated ACSL6 antibodies typically perform optimally at dilutions of 1:1000, though this may vary based on the specific product and application .

• Substrate selection: For analyzing high-abundance ACSL6 expression, chromogenic substrates like DAB (3,3'-diaminobenzidine) provide cost-effective detection . For lower expression levels or quantitative analysis, enhanced chemiluminescent substrates offer superior sensitivity.

• Protein loading controls: When analyzing ACSL6 expression across different experimental conditions, appropriate loading controls are essential. GAPDH antibodies have been validated as effective loading controls at dilutions as high as 1:1000000 .

What are the critical considerations when investigating tissue-specific ACSL6 isoform expression?

Investigating tissue-specific ACSL6 isoform expression presents unique methodological challenges:

• Isoform-specific detection strategies:

  • Western blotting with gradient gels (7.5%) enhances separation of closely migrating ACSL6 isoforms

  • 2D gel electrophoresis may be necessary to distinguish isoforms with similar molecular weights but different isoelectric points

  • Molecular weight considerations: ACSL6 isoforms have been detected at both 75 kDa and 130 kDa ranges

• Subcellular fractionation protocols:

  • Membrane fractions should be prepared by centrifugation at 100,000 g for 60 minutes at 10°C to effectively isolate membrane-associated ACSL6 variants

  • Soluble fractions represent cytosolic ACSL6 populations and should be analyzed separately from membrane fractions

• Tissue-specific expression patterns:

  • Erythroid lineage: Multiple ACSL6 variants have been detected in CD34+ cells, reticulocytes, and K-562 erythroleukemic cells

  • Skeletal muscle: ACSL6 expression is dynamically regulated by nutritional status and exercise

  • Brain tissue: ACSL6 plays a significant role in fatty acid metabolism within neuronal tissue

• PCR-based isoform analysis:

  • Transcript-specific primers targeting unique exon junctions can quantify isoform-specific mRNA levels

  • RACE-PCR approaches have successfully identified novel ACSL6 variants in previous studies

How does ACSL6 function differ from other ACSL family members, and what methodological approaches can distinguish them?

ACSL6 belongs to a family of long-chain acyl-CoA synthetases that includes ACSL1, ACSL3, ACSL4, and ACSL5. While these enzymes catalyze similar reactions, they exhibit distinct tissue distribution, substrate preferences, and cellular functions:

• Functional differentiation:

  • ACSL6 preferentially processes palmitoleate, oleate, and linoleate

  • ACSL1 demonstrates broader substrate specificity and is involved in both lipid synthesis and β-oxidation

  • Cross-reactivity between antibodies targeting different ACSL family members can complicate experimental interpretation

• Methodological approaches for distinguishing ACSL family members:

  a. Antibody selection:

  • Use of highly specific antibodies such as the rabbit recombinant monoclonal antibody that targets specific epitopes of ACSL6

  • Careful validation through knockout/knockdown controls to confirm specificity

  • Consideration of isoform-specific antibodies when investigating specific variants

  b. Expression analysis:

  • ACSL6 mRNA is distinctly regulated by nutritional status in skeletal muscle, with reduced expression during exercise and fasting

  • ACSL1 transcript variants show different distribution patterns than ACSL6 variants in hematopoietic cells (see comparative data below)

c. Functional studies:
- RNA interference targeting specific ACSL family members
- Metabolic flux analysis using isotope-labeled fatty acid substrates
- Subcellular localization studies to identify compartment-specific functions

What are the optimal experimental conditions for using ACSL6 antibody, HRP conjugated in Western blotting?

Western blotting with HRP-conjugated ACSL6 antibodies requires optimization of several parameters:

• Sample preparation:

  • Protein denaturation should be performed at 37°C for 20 minutes to preserve high molecular weight ACSL6 species (130 kDa)

  • Avoid boiling samples or excessive dilution in SDS-PAGE loading buffer, as these conditions may cause loss of detection for certain ACSL6 isoforms

  • For membrane-associated ACSL6 isoforms, ultracentrifugation at 100,000 g for 60 minutes at 10°C provides effective fractionation

• Electrophoresis conditions:

  • 7.5% SDS-PAGE gels offer optimal resolution for ACSL6 proteins in the 68-130 kDa range

  • Load approximately 10 μg of total protein per lane for standard detection

• Transfer conditions:

  • Semi-dry transfer systems with PVDF membranes work effectively for ACSL6 detection

  • Transfer efficiency should be verified using reversible staining methods before immunodetection

• Blocking and antibody incubation:

  • 5% non-fat dry milk in TBST has been validated as an effective blocking agent

  • Optimal dilution for HRP-conjugated ACSL6 antibodies is typically 1:1000

  • Incubation overnight at 4°C may improve signal-to-noise ratio for low-abundance samples

• Detection and visualization:

  • For colorimetric detection, ImmonoPure DAB provides reliable results

  • For enhanced sensitivity, chemiluminescent substrates compatible with HRP should be selected

  • Exposure times should be optimized based on expression levels to avoid signal saturation

What controls should be incorporated when performing experiments with ACSL6 antibody, HRP conjugated?

Rigorous experimental design requires appropriate controls to ensure reliable and interpretable results:

• Positive controls:

  • K562 cell lysates express multiple ACSL6 isoforms and serve as positive controls for antibody validation

  • Recombinant ACSL6 proteins can provide definitive size standards for isoform identification

  • Tissues with known high ACSL6 expression (such as brain tissue) can serve as biological positive controls

• Negative controls:

  • ACSL6 knockdown or knockout samples provide the most stringent specificity controls

  • Primary antibody omission controls help identify non-specific binding of detection reagents

  • Peptide competition assays using the immunizing peptide (such as the C-terminal peptide SGLHSFEQVKAIHIHSDMFSVQNGLLTPTLKAKRPELREYFKKQIEELYS for certain ACSL6 antibodies)

• Loading controls:

  • GAPDH antibodies have been validated for use with ACSL6 detection protocols

  • For membrane fraction analysis, membrane-associated proteins such as Na+/K+-ATPase provide appropriate normalizing controls

  • For cross-species comparisons, consider using evolutionarily conserved housekeeping proteins

• Protocol validation:

  • When analyzing ACSL6 in new experimental systems, perform dilution series to confirm linear detection range

  • Verify antibody reactivity across species if conducting comparative studies (confirmed reactivity includes human, mouse, rat, bovine, canine, guinea pig, horse, rabbit, and zebrafish)

How can researchers troubleshoot common issues in ACSL6 antibody, HRP conjugated experiments?

Troubleshooting guide for common challenges encountered when working with HRP-conjugated ACSL6 antibodies:

• Weak or absent signal:

  • Check protein extraction protocol - ensure membrane proteins are effectively solubilized

  • Verify sample denaturation conditions - avoid boiling which may disrupt detection of high molecular weight ACSL6 species (130 kDa)

  • Increase antibody concentration or extend incubation times

  • Consider using enhanced sensitivity detection substrates

  • Verify antibody storage conditions - HRP-conjugated antibodies should be stored at -20°C or -80°C for long-term storage, 4°C for working solutions

• Multiple unexpected bands:

  • Consider native ACSL6 isoforms - multiple bands at 75 kDa and 130 kDa have been documented in erythroid cells

  • Evaluate potential cross-reactivity with other ACSL family members

  • Increase blocking stringency to reduce non-specific binding

  • Consider using freshly prepared samples to minimize proteolytic degradation

• High background:

  • Optimize blocking conditions - 5% non-fat dry milk in TBST is recommended

  • Increase washing duration and frequency

  • Dilute primary antibody further

  • Reduce substrate incubation time

• Inconsistent results between experiments:

  • Standardize protein extraction and sample preparation protocols

  • Prepare master mixes of antibody dilutions to minimize pipetting errors

  • Use internal controls across experiments for normalization

  • Consider batch effects from different lots of antibodies or reagents

How is ACSL6 antibody being used to investigate metabolic disorders?

Recent research has employed ACSL6 antibodies to elucidate the enzyme's role in various metabolic disorders:

• Skeletal muscle metabolism:

  • ACSL6 mRNA is present in human and rat skeletal muscle, with expression modulated by nutritional status

  • Exercise and fasting decrease ACSL6 mRNA levels, while acute lipid ingestion increases expression

  • ACSL6 gene inhibition in rat primary myotubes decreased lipid accumulation and activated mitochondrial oxidative capacity through the AMPK/PGC1-α pathway

  • ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism

These findings suggest that ACSL6 serves as a metabolic regulator that influences the balance between lipid storage and oxidation in skeletal muscle. Antibody-based detection of ACSL6 protein levels and post-translational modifications has been instrumental in confirming these relationships.

• Brain lipid metabolism:

  • ACSL6 plays a major role in fatty acid metabolism in the brain

  • Alterations in ACSL6 function may contribute to neurological disorders involving disrupted lipid homeostasis

  • Immunohistochemical applications of ACSL6 antibodies help map expression patterns across different brain regions

• Hematological disorders:

  • Translocations between ACSL6 and ETV6 genes are associated with myelodysplastic syndrome with basophilia, acute myelogenous leukemia with eosinophilia, and acute eosinophilic leukemia

  • ACSL6 antibodies provide valuable tools for analyzing expression patterns in patient samples and model systems

What are the considerations for using ACSL6 antibody, HRP conjugated in multiplex immunoassays?

Multiplex immunoassays allow simultaneous detection of multiple proteins, offering advantages for comprehensive analysis of metabolic pathways involving ACSL6:

• Antibody compatibility:

  • HRP-conjugated ACSL6 antibodies must be compatible with other detection systems used in multiplex assays

  • Consider using spectrally distinct substrates for different HRP-conjugated antibodies

  • Tyramide signal amplification systems can enhance sensitivity and multiplexing capability

• Sequential detection protocols:

  • When using multiple HRP-conjugated antibodies, complete stripping between detection cycles is essential

  • Validate stripping efficiency by re-probing with secondary antibodies alone

  • Consider size differences between target proteins for multiplexing on the same membrane

• Alternative conjugation options:

  • For fluorescence-based multiplex systems, ACSL6 antibodies can be custom conjugated with various fluorophores including:

    • Alexa Fluor dyes (AF350, AF488, AF555, AF594, AF647, AF680, AF700, AF750)

    • iFluor dyes (350-860 nm range)

    • mFluor dyes (UV375-Red 780)

  • Tandem dyes for flow cytometry applications (APC, PE, PerCP and their tandems)

• Cross-platform validation:

  • Confirm results from multiplex assays with single-plex detection systems

  • Evaluate potential signal interference between detection systems

  • Establish appropriate controls for each target in the multiplex panel

How might advances in ACSL6 antibody technology contribute to understanding metabolic diseases?

The continued refinement of ACSL6 antibody technology will likely accelerate research in several directions:

• Isoform-specific antibodies:

  • Development of antibodies that specifically recognize different ACSL6 splice variants

  • Implementation in tissue-specific metabolic studies to determine functional specialization

  • Investigation of differential regulation in pathological conditions

• Post-translational modification mapping:

  • Phospho-specific ACSL6 antibodies to investigate regulatory mechanisms

  • Antibodies recognizing other modifications (acetylation, ubiquitination) to study protein stability and turnover

  • Correlation of modifications with enzymatic activity in different metabolic states

• High-resolution imaging applications:

  • Super-resolution microscopy using fluorescently-conjugated ACSL6 antibodies

  • Co-localization studies with other metabolic enzymes and organelle markers

  • Live-cell imaging with cell-permeable antibody derivatives

• Precision medicine applications:

  • Diagnostic tools for hematological malignancies involving ACSL6-ETV6 translocations

  • Stratification markers for metabolic disorders with altered lipid metabolism

  • Companion diagnostics for emerging therapeutics targeting lipid metabolic pathways

What methodological advances might improve ACSL6 detection sensitivity and specificity?

Several emerging technologies hold promise for enhancing ACSL6 detection:

• Proximity ligation assays:

  • Detection of protein-protein interactions involving ACSL6

  • Increased sensitivity through signal amplification

  • Analysis of ACSL6 associations with other metabolic enzymes

• Single-molecule detection methods:

  • Digital ELISA platforms for ultrasensitive ACSL6 quantification

  • Single-cell western blotting for heterogeneity analysis

  • Correlation with functional metabolic parameters

• Mass spectrometry-based immunoprecipitation:

  • Coupling ACSL6 antibodies with mass spectrometry for detailed proteomic analysis

  • Identification of novel interaction partners

  • Characterization of post-translational modifications

• CRISPR-based tagging strategies:

  • Endogenous tagging of ACSL6 for improved detection specificity

  • Live-cell tracking of ACSL6 dynamics

  • Correlation of localization with functional studies