ACSL3 Antibody

What is ACSL3 and what is its biological function?

ACSL3 (Acyl-CoA Synthetase Long-Chain Family Member 3) is a member of the acyl-CoA synthetase family that plays a crucial role in lipid metabolism. This enzyme catalyzes the conversion of long-chain fatty acids into acyl-CoA, which is essential for the synthesis of cellular lipids and the β-oxidation pathway. This enzymatic activity is vital for energy production and the formation of key lipid molecules such as triacylglycerols, phospholipids, and cholesteryl esters . ACSL3 is particularly important in tissues with high metabolic demands, functioning primarily in an anabolic role in energy metabolism. It has been shown to mediate hepatic lipogenesis and is required for the incorporation of fatty acids into phosphatidylcholine, the major phospholipid located on the surface of VLDL (very low density lipoproteins) .

ACSL3 exhibits substrate specificity, preferentially utilizing myristate, laurate, arachidonate, and eicosapentaenoate as substrates, which distinguishes it from other family members like ACSL1 . This unique substrate preference contributes to its specialized physiological roles in different tissues.

What types of ACSL3 antibodies are available for research applications?

Several types of ACSL3 antibodies are available for research, varying in host species, clonality, and target epitopes:

These antibodies target different regions of the ACSL3 protein, providing researchers with options based on their specific experimental requirements and target species .

How should I select the most appropriate ACSL3 antibody for my research application?

When selecting an ACSL3 antibody for your research, consider the following methodological approach:

• Determine your experimental application: Different antibodies perform optimally in specific applications. For example, if you're conducting Western blotting, immunohistochemistry, or immunofluorescence, review validation data for each antibody in your application of interest. The H-9 monoclonal antibody has been validated for WB, IP, IF, IHC, and ELISA , while some polyclonal antibodies may have more specific application profiles.

• Species reactivity considerations: Confirm that the antibody recognizes ACSL3 from your model organism. For cross-species studies, select antibodies with demonstrated reactivity across your species of interest. For instance, some antibodies show reactivity with human, mouse, and rat samples, while others may have more limited species reactivity .

• Epitope specificity analysis: Consider which region of the ACSL3 protein you need to target. Antibodies target different regions (N-terminal, C-terminal, or internal domains), which may affect their functionality in certain applications or when studying specific isoforms .

• Clonality requirements: Determine whether a monoclonal or polyclonal antibody better suits your needs:

  • Monoclonal antibodies (like H-9) offer high specificity but recognize a single epitope

  • Polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals but with potential for increased background

• Validation extent review: Examine the validation data provided by manufacturers, paying particular attention to:

  • Published citations using the antibody

  • Specific validation techniques employed (knockout validation is particularly valuable)

  • Molecular weight confirmation (ACSL3 is typically observed at 70-80 kDa)

For quantitative applications requiring precise measurements of ACSL3 levels, monoclonal or recombinant antibodies often provide better reproducibility and specificity .

What are the optimal protocols for using ACSL3 antibodies in Western blotting applications?

For optimal Western blotting with ACSL3 antibodies, follow these methodological guidelines:

• Sample preparation optimization:

  • For cellular samples: Use validated cell lysis buffers compatible with membrane proteins

  • For tissue samples: Homogenize thoroughly in appropriate buffer with protease inhibitors

  • Protein concentration should be determined and standardized (15-30 μg total protein per lane is typically sufficient)

• Gel electrophoresis considerations:

  • Use 7.5-10% SDS-PAGE gels for optimal resolution of ACSL3 (approximately 70-80 kDa)

  • Include molecular weight markers that span the range around 80 kDa

• Transfer and blocking optimization:

  • For ACSL3, semi-dry or wet transfer protocols are both effective

  • 5% non-fat dry milk in TBST is often recommended as blocking buffer

  • Some antibodies may perform better with specific blocking agents (check manufacturer recommendations)

• Antibody dilution and incubation parameters:

  • Primary antibody dilutions vary by product:

    • ACSL3 Antibody (20710-1-AP): 1:2000-1:12000

    • ACSL3 Antibody (30214-1-AP): 1:1000-1:8000

  • Incubate at 4°C overnight for optimal signal-to-noise ratio

  • Use appropriate species-specific secondary antibody conjugated to HRP or fluorescent labels

• Detection and visualization techniques:

  • ECL Plus detection systems provide good sensitivity for ACSL3

  • For challenging samples, consider using signal enhancement methods

  • ACSL3 appears as a band at approximately 70-80 kDa (specific molecular weight may vary slightly by antibody and sample type)

• Validation controls integration:

  • Include positive control samples (HEK-293, LNCaP, HuH-7 cells show reliable ACSL3 expression)

  • When possible, include ACSL3 knockout or knockdown samples as negative controls

How can I use ACSL3 antibodies to investigate tissue-specific expression patterns?

To effectively investigate tissue-specific ACSL3 expression patterns, employ this comprehensive methodological approach:

• Tissue sample selection and preparation strategy:

  • Target relevant tissues based on known ACSL3 expression profile: ACSL3 shows high expression in brain and testis, moderate in liver and adipose tissue, low in muscle, and minimal in heart

  • Prepare tissues using fixation protocols optimized for membrane protein preservation

  • For comparative studies, standardize collection and processing across all tissue types

• Immunohistochemistry protocol optimization:

  • Antigen retrieval is critical: For ACSL3, TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 can be an alternative

  • Dilution ranges: 1:50-1:500 for IHC applications with polyclonal antibodies like 20710-1-AP

  • Include positive control tissues (e.g., human stomach cancer tissue has been validated)

• Immunofluorescence alternatives for colocalization studies:

  • Use ACSL3 antibodies validated for IF applications, such as the H-9 monoclonal antibody

  • Consider dual-staining with organelle markers (particularly lipid droplet markers) to determine subcellular localization

  • ACSL3 has been found in lipid droplet fractions in human hepatocyte HuH7 cells and stimulated 3T3-L1 adipocytes

• Quantitative analysis implementation:

  • Develop standardized scoring systems for IHC intensity

  • For IF, employ digital image analysis with appropriate controls for background subtraction

  • Western blot analysis can complement IHC data for quantitative comparisons across tissues

• Validation through complementary techniques:

  • Correlate protein expression with mRNA levels using RT-qPCR

  • Consider in situ hybridization to confirm tissue-specific expression patterns

  • When making cross-species comparisons, acknowledge potential differences in expression patterns between species

Early studies demonstrated that ACSL3's tissue distribution differs from other ACSL family members, with particularly high expression in brain and testis. This unique expression pattern suggests specialized functions in these tissues that can be further explored through careful immunohistochemical analysis .

What strategies can resolve common issues with ACSL3 antibody specificity and cross-reactivity?

When encountering specificity or cross-reactivity issues with ACSL3 antibodies, implement these advanced troubleshooting strategies:

• Cross-reactivity assessment with ACSL family members:

  • ACSL3 shares sequence homology with other ACSL family members

  • Validate antibody specificity using overexpression systems of individual ACSL isoforms

  • Consider using cell lines with CRISPR/Cas9 knockout of ACSL3 as negative controls

• Epitope mapping and blocking peptide validation:

  • Determine if your antibody targets conserved or unique regions of ACSL3

  • For polyclonal antibodies, use blocking peptides corresponding to the immunogen to confirm specificity

  • Different antibodies target distinct regions (N-terminal, C-terminal, internal domains), affecting their specificity profiles

• Multiple antibody approach for validation:

  • Use two different antibodies targeting distinct epitopes of ACSL3

  • Compare detection patterns: consistent results across antibodies increase confidence in specificity

  • Example combination: Use both rabbit polyclonal (e.g., ab151959) and mouse monoclonal (e.g., H-9) antibodies

• Optimization of experimental conditions:

  • Adjust antibody concentration: Titrate to find optimal signal-to-noise ratio

  • Modify blocking conditions: Test alternative blocking agents if background is high

  • Increase stringency of washing steps to reduce non-specific binding

• Lysate preparation considerations:

  • ACSL3 is a membrane-associated protein; use detergent-based lysis buffers

  • Include appropriate protease inhibitors to prevent degradation

  • Fresh sample preparation may yield better results than frozen samples

• Western blot troubleshooting details:

  • Multiple bands: May indicate isoforms, degradation products, or post-translational modifications

  • No signal: Verify sample expression levels (HEK-293, LNCaP, HuH-7 cells show reliable ACSL3 expression)

  • High background: Optimize blocking and antibody concentrations

When analyzing Western blot results, note that the observed molecular weight of ACSL3 can range from 70-80 kDa, with some antibodies specifically detecting it at approximately 72 kDa (30214-1-AP) or 70-80 kDa (20710-1-AP) .

How can ACSL3 antibodies be utilized in studies of lipid metabolism disorders?

For investigating ACSL3's role in lipid metabolism disorders, implement these specialized methodological approaches:

• Tissue-specific expression analysis in pathological states:

  • Compare ACSL3 expression between normal and diseased tissues using immunohistochemistry

  • Quantify changes in ACSL3 protein levels via Western blot in disease models

  • Focus on tissues with known ACSL3 expression and metabolic significance: liver, adipose tissue, brain, and testis

• Subcellular localization studies in metabolic disease states:

  • Use immunofluorescence with ACSL3 antibodies to track potential changes in subcellular distribution

  • Co-localization with lipid droplet markers is particularly informative, as ACSL3 has been found in lipid droplet fractions

  • Changes in localization may indicate altered function in disease states

• Functional activity correlation with expression:

  • Complement antibody-based detection with ACSL activity assays

  • The methodology described in PMC2878065 can be adapted to measure ACSL3-specific activity:

    • Treat cells with specific conditions (e.g., L165041 treatment)

    • Prepare cell lysates under standardized conditions

    • Conduct activity assays using ACSL3's preferred substrates

• Intervention studies with gene modulation:

  • Use ACSL3 antibodies to validate knockdown or overexpression in experimental models

  • Previous research demonstrated that depletion of ACSL3 by specific siRNA transfection abolished the effects of OSM (oncostatin M) on FA metabolism

  • Monitor changes in ACSL3 protein levels in response to therapeutic interventions

• PPARδ-ACSL3 pathway investigation:

  • Research has shown that ACSL3 transcription is regulated by PPARδ

  • Use ACSL3 antibodies in conjunction with PPARδ agonists (like L165041) to monitor corresponding changes in ACSL3 protein levels

  • This pathway is particularly relevant in hepatic lipid metabolism disorders

Previous research has established connections between ACSL3 expression changes and altered lipid metabolism. For example, OSM treatment resulted in increased ACSL3 expression associated with decreased cellular triglyceride content and enhanced fatty acid β-oxidation in hepatic cells . Similarly, feeding hamsters with a fat- and cholesterol-enriched diet specifically increased ACSL3 mRNA and protein expression in liver tissue .

What are the advanced techniques for studying ACSL3's role in fatty acid metabolism using immunoprecipitation?

To effectively study ACSL3's role in fatty acid metabolism using immunoprecipitation (IP), implement these advanced methodological approaches:

• Selection of optimal IP-validated antibodies:

  • Use antibodies specifically validated for IP applications

  • Examples include ACSL3 Antibody (H-9) from Santa Cruz Biotechnology and ACSL3 antibody (30214-1-AP) from Proteintech

  • For 30214-1-AP, recommended IP dilution is 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Sample preparation optimization for membrane-associated proteins:

  • ACSL3 is associated with membranes and lipid droplets

  • Use non-denaturing lysis buffers containing mild detergents (e.g., 1% NP-40 or 0.5% Triton X-100)

  • Include protease and phosphatase inhibitors to maintain protein integrity and modification state

  • Cell types with validated IP protocols include LNCaP cells

• Co-immunoprecipitation (Co-IP) for protein interaction studies:

  • Identify ACSL3 binding partners involved in fatty acid metabolism

  • Co-IP followed by mass spectrometry can reveal novel interaction networks

  • Validate interactions through reciprocal IP and Western blotting

  • Consider native IP conditions to preserve protein complexes

• Functional analysis of immunoprecipitated ACSL3:

  • Assess enzymatic activity of immunoprecipitated ACSL3 using acyl-CoA synthetase activity assays

  • Compare activities with different fatty acid substrates (laurate, myristate, arachidonate, eicosapentaenoate)

  • Analyze post-translational modifications that may regulate activity

• Integration with metabolic labeling techniques:

  • Combine IP with metabolic labeling using radioactive or stable isotope-labeled fatty acids

  • Track incorporation of labeled fatty acids into lipid species

  • Compare wild-type ACSL3 with mutant forms to determine structure-function relationships

• IP-based chromatin immunoprecipitation (ChIP) for transcriptional studies:

  • Research has identified PPARδ as a regulator of ACSL3 gene expression

  • ChIP assays with PPARδ antibodies can identify direct binding to ACSL3 promoter regions

  • This approach helps elucidate transcriptional regulation mechanisms of ACSL3

Research has demonstrated that ACSL3 preferentially utilizes specific fatty acids like laurate, myristate, arachidonate, and eicosapentaenoate, distinguishing it from other ACSL family members . IP-based studies can further clarify how this substrate specificity contributes to ACSL3's unique roles in cellular lipid metabolism.

How are ACSL3 antibodies being utilized in cancer research, and what methodological considerations apply?

ACSL3 antibodies are becoming increasingly important in cancer research, with these methodological considerations for implementation:

• Differential expression analysis in tumor vs. normal tissues:

  • Use immunohistochemistry with ACSL3 antibodies to compare expression patterns

  • Recommended antibodies include polyclonal options like 20710-1-AP (dilution 1:50-1:500)

  • Antigen retrieval optimization is critical: TE buffer pH 9.0 or citrate buffer pH 6.0 have been validated

  • Human stomach cancer tissue has been used as a positive control for IHC applications

• Cell line model validation and characterization:

  • Western blot analysis of ACSL3 expression across cancer cell line panels

  • Several cancer cell lines have been validated for ACSL3 detection:

    • LNCaP (prostate cancer)

    • HuH-7 (hepatocellular carcinoma)

    • DU 145 (prostate cancer)

    • MKN-45 (gastric cancer)

• Correlation with fatty acid metabolism alterations in cancer:

  • ACSL3 catalyzes the conversion of long-chain fatty acids to acyl-CoA, a critical step in both lipid synthesis and β-oxidation

  • Cancer cells often exhibit altered lipid metabolism; ACSL3 may play a role in supporting increased lipid synthesis in rapidly proliferating cells

  • Combine ACSL3 expression analysis with metabolic profiling of fatty acid utilization

• Regulation by oncogenic signaling pathways:

  • Previous research showed that ACSL3 is regulated by oncostatin M (OSM)

  • Investigate relationships between ACSL3 expression and other oncogenic pathways

  • Use phospho-specific antibodies to examine potential post-translational regulation

• Functional studies using genetic manipulation:

  • Validate knockdown or overexpression of ACSL3 using antibody detection

  • Assess phenotypic consequences on cancer cell proliferation, migration, and metabolism

  • Correlate changes in ACSL3 protein levels with alterations in specific lipid species

Emerging research suggests that ACSL3 may play important roles in cancer metabolism, particularly in contexts where fatty acid utilization is altered. The connection between ACSL3 expression and the PPARδ pathway is particularly interesting, as PPARs are known to regulate genes involved in cancer metabolism and progression.

What are the current challenges and emerging techniques in detecting post-translational modifications of ACSL3?

Investigating post-translational modifications (PTMs) of ACSL3 presents unique challenges and opportunities, with these methodological considerations:

• Phosphorylation detection strategies:

  • Standard approach: Immunoprecipitate ACSL3 using validated antibodies (e.g., H-9 or 30214-1-AP)

  • Analyze by Western blotting with phospho-specific antibodies

  • For unbiased discovery: IP followed by mass spectrometry phosphopeptide enrichment

  • Challenge: Limited availability of phospho-specific ACSL3 antibodies necessitates alternative approaches

• Ubiquitination and SUMOylation analysis techniques:

  • Denaturing IP protocols (with SDS in lysis buffer) are essential to disrupt associated proteins

  • Detect with ubiquitin or SUMO-specific antibodies after ACSL3 immunoprecipitation

  • Alternative: Express tagged ubiquitin/SUMO constructs and detect modification of immunoprecipitated ACSL3

  • These modifications may regulate ACSL3 stability and subcellular localization

• Acetylation and other PTM detection methodologies:

  • IP-mass spectrometry approaches for unbiased PTM profiling

  • Western blotting with pan-acetyl-lysine antibodies after ACSL3 immunoprecipitation

  • Correlation with histone deacetylase (HDAC) or sirtuin activity/inhibition

  • Acetylation may regulate ACSL3 enzymatic activity and substrate preference

• PTM-specific antibody development considerations:

  • Current limitation: Lack of commercially available PTM-specific ACSL3 antibodies

  • Future direction: Development of antibodies targeting key modified residues

  • Validation strategies will require phosphatase/deubiquitinase treatments as controls

  • Synthetic peptide competition assays to confirm specificity

• Functional impact assessment strategies:

  • Correlate PTM status with enzymatic activity measurements

  • Site-directed mutagenesis of modified residues to mimic or prevent modification

  • Subcellular localization studies under conditions that induce or inhibit specific PTMs

  • Association with regulatory proteins that may be influenced by PTM status

A key challenge in the field is connecting specific PTMs to functional outcomes. For example, ACSL3's roles in lipid metabolism, including its preference for certain fatty acid substrates (laurate, myristate, arachidonate, eicosapentaenoate) , may be dynamically regulated by PTMs in response to metabolic conditions. Advanced IP-MS techniques combined with functional assays will be essential to unravel these regulatory mechanisms.

What are the most reliable positive control samples and validation approaches for ACSL3 antibody experiments?

Based on extensive research literature, these methodological recommendations ensure reliable ACSL3 antibody validation:

• Cellular positive controls with verified expression:

  • Human cell lines: HEK-293, LNCaP, HuH-7, and DU 145 cells consistently show detectable ACSL3 expression

  • These cell lines have been validated across multiple antibodies and can serve as reliable positive controls

  • For mouse studies: kidney tissue has been confirmed as a positive control

• Tissue positive controls for immunohistochemistry:

  • Human samples: Human stomach cancer tissue has been validated for IHC applications

  • Animal models: Brain and testis tissues show highest expression, with moderate expression in liver and adipose tissue

  • Antigen retrieval optimization is essential: TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative

• Genetic validation approaches:

  • CRISPR/Cas9 knockout controls: Compare staining in wild-type versus ACSL3-knockout cell lines

  • siRNA or shRNA knockdown validation: Demonstrate reduced antibody signal corresponding to reduced ACSL3 levels

  • Overexpression validation: Show increased signal intensity in cells transfected with ACSL3 expression constructs

• Cross-antibody validation strategy:

  • Use multiple antibodies targeting different epitopes of ACSL3:

    • Monoclonal antibody (H-9) targeting mouse, rat, and human ACSL3

    • Polyclonal antibody (ab151959) targeting region within aa 400-650

    • Recombinant monoclonal antibody [EPR29199-39] for high specificity

  • Consistent results across antibodies significantly increase confidence in specificity

• Molecular weight verification:

  • Expected molecular weight range: 70-80 kDa

  • Specific observations: 72 kDa (30214-1-AP) or 70-80 kDa (20710-1-AP)

  • Include appropriate molecular weight markers and positive control lysates

This systematic approach to validation ensures reliable and reproducible results when working with ACSL3 antibodies across different experimental applications and model systems.

How can researchers integrate ACSL3 antibody-based detection with other methodologies to gain comprehensive insights into lipid metabolism?

To achieve comprehensive understanding of ACSL3's role in lipid metabolism, integrate antibody-based detection with these complementary methodologies:

• Multi-omics integration strategy:

  • Combine ACSL3 protein quantification (antibody-based) with:

    • Transcriptomics: RNA-Seq or qPCR to correlate protein with mRNA levels

    • Lipidomics: MS-based profiling of fatty acid and lipid species

    • Metabolomics: Analysis of metabolic intermediates in fatty acid pathways

  • This approach provides a systems-level view of how ACSL3 expression impacts the lipidome

• Enzyme activity correlation methodology:

  • Pair Western blot quantification of ACSL3 with acyl-CoA synthetase activity assays

  • Methodology described in PMC2878065 can be adapted:

    • Prepare cell lysates under standardized conditions

    • Measure activity using ACSL3's preferred substrates (laurate, myristate, arachidonate, eicosapentaenoate)

  • Correlate protein levels with activity to identify potential post-translational regulation

• Imaging technologies integration:

  • Combine immunofluorescence with:

    • Lipid droplet staining (e.g., BODIPY or Oil Red O)

    • Live-cell imaging of fluorescently labeled fatty acids

    • Super-resolution microscopy for precise subcellular localization

  • This reveals ACSL3's dynamic localization in relation to lipid metabolism

• Genetic manipulation with phenotypic readouts:

  • Use CRISPR/Cas9 to generate ACSL3 knockouts or specific mutations

  • Validate using antibody detection

  • Assess phenotypic consequences:

    • Changes in lipid droplet formation and size

    • Alterations in specific lipid species synthesis

    • Effects on cellular energetics and growth

• Pathway analysis with pharmacological modulation:

  • Previous research established that PPARδ regulates ACSL3 expression

  • Use PPARδ agonists (e.g., L165041) or antagonists while monitoring ACSL3 protein levels

  • This approach reveals regulatory mechanisms and potential therapeutic targets

  • Complement with ChIP assays to confirm direct transcriptional regulation