ACSL5 Antibody

What is ACSL5 and what cellular functions does it perform?

ACSL5 (acyl-CoA synthetase long chain family member 5) is a 76 kDa protein comprising 683 amino acid residues in humans. As a member of the ATP-dependent AMP-binding enzyme family, ACSL5 catalyzes the conversion of long-chain fatty acids to their active form (acyl-CoAs), which are essential for both cellular lipid synthesis and degradation via beta-oxidation. The protein localizes to the mitochondria, endoplasmic reticulum, and cell membrane, with up to three different isoforms reported. ACSL5 is prominently expressed in various tissues, particularly in the colon and duodenum . Recent research has revealed its regulatory role in cancer processes, specifically in the anti-tumor function of palmitic acid (C16:0) in non-small lung cancer cells .

How should I select an appropriate ACSL5 antibody for my specific research application?

When selecting an ACSL5 antibody, consider the following methodological approach:

• Application compatibility: Determine which applications you need (Western blot, IHC, IF, ELISA) and select antibodies validated for those specific techniques. For instance, if performing Western blot analysis, ensure the antibody has been validated for this application with published results .

• Species reactivity: Verify the antibody's reactivity with your species of interest. Many ACSL5 antibodies are reactive with human, mouse, and rat samples, but reactivity can vary between products .

• Target epitope: Consider whether you need an antibody targeting a specific region of ACSL5 (N-terminal, C-terminal, or internal domain). This is particularly important if studying specific isoforms or if post-translational modifications might affect antibody binding .

• Validation data: Prioritize antibodies with published citations and validation data specific to your experimental system. For example, some ACSL5 antibodies have been validated in non-small cell lung cancer research .

• Clone type: Determine whether a monoclonal or polyclonal antibody is more suitable for your research question. Monoclonal antibodies offer higher specificity but may be more sensitive to epitope changes, while polyclonals provide broader antigen recognition .

What are the most common applications for ACSL5 antibodies in research settings?

ACSL5 antibodies are employed across various research applications, with Western blot and immunohistochemistry being the most widely utilized techniques. Other common applications include:

• Western Blot (WB): Widely used for detecting and quantifying ACSL5 protein expression levels in cell and tissue lysates. This technique allows researchers to identify specific isoforms and evaluate expression changes under different experimental conditions .

• Immunohistochemistry (IHC): Applied to assess ACSL5 localization and expression patterns in tissue sections, which is particularly valuable in cancer research to correlate expression with pathological features .

• Immunofluorescence (IF) and Immunocytochemistry (ICC): Used to visualize subcellular localization of ACSL5 in cultured cells and tissue sections .

• Immunoprecipitation (IP): Employed to isolate ACSL5 protein complexes to study protein-protein interactions .

• ELISA: Utilized for quantitative measurement of ACSL5 in biological samples .

• Flow Cytometry (FCM): Used for analyzing ACSL5 expression at the single-cell level .

Recent research has demonstrated the application of ACSL5 antibodies in evaluating expression changes following treatment with palmitic acid (C16:0) in cancer cells, highlighting their utility in mechanistic studies of lipid metabolism and signaling pathways .

What is the optimal protocol for using ACSL5 antibodies in Western blot analysis?

For optimal Western blot detection of ACSL5, follow this methodological approach:

Sample Preparation:

• Prepare cell or tissue lysates using RIPA lysis buffer supplemented with protease inhibitors (as used in A549 cell studies) .

• Determine protein concentration using spectrophotometric methods (e.g., measuring absorbance at 260 nm with a NanoDrop instrument) .

• Load 20 μg of protein per lane on SDS-PAGE gels .

Blotting and Detection:

• Separate proteins using SDS-PAGE and transfer to PVDF membrane.

• Block the membrane with blocking solution for 15 minutes at room temperature with gentle shaking .

• Incubate with primary anti-ACSL5 antibody (typically at 1:1000 dilution) overnight at 4°C .

• Wash the membrane thoroughly with TBST buffer.

• Incubate with appropriate HRP-conjugated secondary antibody.

• Develop using chemiluminescence detection reagents.

• Quantify band intensity using image analysis software such as ImageJ .

Critical Considerations:

• ACSL5 appears at approximately 76 kDa on Western blots.

• Include appropriate positive controls and loading controls (β-actin is commonly used).

• When comparing ACSL5 expression between experimental conditions (e.g., C16:0 treatment), maintain consistent protein loading and exposure times .

What are the best practices for ACSL5 antibody use in immunohistochemistry?

For optimal immunohistochemical detection of ACSL5, follow these methodological steps:

Tissue Processing and Preparation:

• Fix tissue samples appropriately (commonly using formalin) and embed in paraffin.

• Section tissues at 4-5 μm thickness and mount on positively charged slides.

• Deparaffinize sections using xylene and rehydrate through graded alcohols.

Staining Protocol:

• Perform antigen retrieval using sodium citrate buffer (pH 6.0) in an autoclave for 5 minutes .

• Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 minutes .

• Apply primary anti-ACSL5 antibody at appropriate dilution (typically 1:100) and incubate overnight at 4°C .

• Include negative controls (slides not treated with primary antibody).

• Apply biotinylated secondary antibody (dilution 1:200) and incubate for 45 minutes at room temperature .

• Develop with DAB solution and counterstain with hematoxylin for 1 minute .

• Mount slides with coverslips after appropriate clearing.

Quantification:

• Capture digital images of stained sections.

• Quantify immunopositivity using ImageJ or similar image analysis software .

• Analyze expression patterns in relation to tissue morphology and other markers of interest (such as Ki-67 for proliferation or p53 for apoptosis) .

How can I verify the specificity of my ACSL5 antibody?

Verifying antibody specificity is crucial for reliable experimental results. Implement these methodological approaches:

• Positive and negative tissue controls: Test the antibody on tissues known to express high levels of ACSL5 (e.g., colon, duodenum) and those with minimal expression .

• RNA interference validation: Perform siRNA knockdown of ACSL5 and confirm reduced antibody signal in Western blots or immunostaining. This approach was successfully used in A549 cells using siRNA sequences targeting ACSL5 :

  • ACSL5-Homo-731: GCUUGUUACACGUACUCUATT, UAGAGUACGUGUAACAAGCTT

  • Transfect cells using lipofectamine 2000 and confirm knockdown efficiency using qRT-PCR .

• Peptide competition assay: Pre-incubate the antibody with a blocking peptide containing the target epitope before application to samples. Signal reduction confirms specificity.

• Multiple antibody validation: Compare staining patterns using different antibodies targeting distinct ACSL5 epitopes.

• Western blot analysis: Verify that the antibody detects a band of the expected molecular weight (76 kDa for ACSL5) .

• Cross-species reactivity: Test the antibody in samples from different species where ACSL5 is conserved (mouse, rat, etc.) to confirm evolutionary conservation of the epitope .

How can ACSL5 antibodies be utilized to investigate lipid metabolism pathways?

ACSL5 antibodies can be strategically employed to elucidate lipid metabolism pathways through these methodological approaches:

• Co-localization studies: Use dual immunofluorescence with ACSL5 antibodies and markers for mitochondria, ER, or lipid droplets to visualize the subcellular compartmentalization of fatty acid activation processes .

• Expression correlation analysis: Combine ACSL5 antibody staining with detection of other lipid metabolism enzymes to establish pathway relationships and regulatory networks.

• Metabolic challenge experiments: Apply ACSL5 antibodies to detect expression changes following challenges with different fatty acid substrates (e.g., palmitic acid), fasting conditions, or metabolic inhibitors .

• Tissue-specific expression profiling: Utilize ACSL5 antibodies to map expression patterns across different tissues with varying metabolic profiles, particularly focusing on tissues with high lipid metabolism rates such as liver, adipose tissue, intestine, and muscle .

• Pathological state assessment: Employ ACSL5 antibodies to evaluate expression changes in disease models associated with dysregulated lipid metabolism, such as obesity, diabetes, non-alcoholic fatty liver disease, and cancer .

• Regulatory mechanism studies: Combine ACSL5 immunodetection with analysis of transcription factors and signaling molecules (e.g., ERK pathway components) to decipher the regulatory mechanisms governing ACSL5 expression and activity in different metabolic states .

How should I design ACSL5 knockdown experiments to study its functional role?

Designing robust ACSL5 knockdown experiments requires careful methodological consideration:

• siRNA design and validation:

  • Design multiple siRNA sequences targeting different regions of ACSL5 mRNA. For example, in A549 cells research, three distinct sequences were tested:

    • ACSL5-Homo-1604: GCUUAUGGUCAAACAGAAUTT, AUUCUGUUUGACCAUAAGCTT

    • ACSL5-Homo-1509: GCGGAAGGGUUCGUGUAAUTT, AUUACACGAACCCUUCCGCTT

    • ACSL5-Homo-731: GCUUGUUACACGUACUCUATT, UAGAGUACGUGUAACAAGCTT

  • Validate knockdown efficiency by qRT-PCR using specific primers:

    • Forward: 5′-GTCATCTGCTTCACCAGTGG-3′

    • Reverse: 5′-CGTCAGCCAGCAACCGAATATCC-3′

  • Use the 2^(-ΔΔCT) method normalized to internal control (e.g., β-actin) to quantify knockdown efficiency .

• Transfection optimization:

  • For A549 cells, use 20 pmol/μL siRNA with Lipofectamine 2000, incubating the mixture at room temperature for 20 minutes before adding to cells .

  • Assess transfection efficiency after 24 hours at 37°C .

• Experimental design:

  • Include appropriate controls: non-targeting siRNA control, mock transfection, and untransfected cells.

  • For rescue experiments, include a condition with siRNA-resistant ACSL5 overexpression.

  • Design time-course experiments to capture both early and late consequences of ACSL5 depletion.

• Functional assessment:

  • Evaluate cellular phenotypes using assays relevant to ACSL5 function:

    • Proliferation: CCK-8 assay

    • Apoptosis: Annexin V-FITC/PI double staining

    • Migration: Wound healing assay

    • Invasion: Transwell assay

  • Analyze lipid metabolism parameters: fatty acid uptake, acyl-CoA synthesis rates, β-oxidation, lipid droplet formation.

  • Examine signaling pathway alterations, particularly the ERK pathway implicated in ACSL5-mediated effects in cancer cells .

• Protein level confirmation:

  • Verify knockdown at the protein level using Western blot with validated ACSL5 antibodies .

  • Quantify band intensity to determine the degree of protein reduction.

What techniques can be used to study ACSL5's role in cancer development?

Investigating ACSL5's role in cancer development requires multiple complementary techniques:

• Expression analysis in clinical samples:

  • Use immunohistochemistry with validated ACSL5 antibodies to compare expression between tumor and adjacent normal tissues .

  • Correlate expression levels with clinical parameters, disease stage, and patient outcomes.

  • Analyze ACSL5 expression in conjunction with proliferation markers (Ki-67) and tumor suppressor proteins (p53) .

• In vitro functional studies:

  • Manipulate ACSL5 expression (knockdown or overexpression) in cancer cell lines.

  • Assess effects on cancer hallmarks:

    • Proliferation using CCK-8 assay

    • Apoptosis using Annexin V-FITC/PI staining

    • Migration using wound healing assay

    • Invasion using transwell assay

  • Evaluate metabolic reprogramming through measurement of fatty acid uptake, utilization, and lipid profiles.

• Signaling pathway analysis:

  • Investigate ACSL5's interaction with established oncogenic pathways, particularly the ERK signaling pathway .

  • Use Western blotting to assess phosphorylation status of ERK after ACSL5 modulation .

  • Employ pathway inhibitors to determine whether ACSL5's effects are dependent on specific signaling cascades.

• In vivo tumor models:

  • Establish xenograft models using cells with modified ACSL5 expression.

  • Administer treatments such as palmitic acid (C16:0) to evaluate effects on tumor growth .

  • Measure tumor volume and perform immunohistochemical analysis of resected tumors .

  • Assess the expression of ACSL5, Ki-67, and p53 in tumor tissues to correlate molecular changes with tumor growth characteristics .

• Metabolite profiling:

  • Analyze changes in lipid metabolites following ACSL5 modulation using mass spectrometry.

  • Correlate metabolic alterations with phenotypic changes in cancer cells.

How can I resolve inconsistent results when using ACSL5 antibodies?

When encountering inconsistent results with ACSL5 antibodies, implement this systematic troubleshooting approach:

• Antibody validation:

  • Verify antibody specificity using positive controls (tissues known to express high levels of ACSL5) and negative controls (ACSL5 knockdown samples) .

  • Test multiple antibodies targeting different epitopes of ACSL5 to confirm results.

  • Check literature for validated antibody clones with consistent performance in your specific application .

• Sample preparation optimization:

  • For Western blot: Ensure complete protein denaturation and use fresh protease inhibitors in lysis buffers .

  • For IHC: Optimize fixation time and antigen retrieval conditions (sodium citrate buffer, pH 6.0, in autoclave for 5 minutes has been effective) .

  • Standardize protein quantification methods to ensure consistent loading .

• Protocol standardization:

  • Maintain consistent antibody concentrations across experiments (1:1000 for Western blot, 1:100 for IHC) .

  • Standardize incubation times and temperatures (overnight at 4°C for primary antibody) .

  • Use automated systems where possible to reduce technical variability.

• Technical considerations:

  • For Western blots: Test different membrane types (PVDF vs. nitrocellulose) and blocking reagents.

  • For IHC: Compare different detection systems and counterstaining protocols.

  • Consider lot-to-lot antibody variability; maintain records of antibody lots used.

• Biological variability assessment:

  • Account for ACSL5 isoform expression differences in different tissues .

  • Consider cell-type specific expression patterns and subcellular localization (mitochondria, ER, cell membrane) .

  • Evaluate expression changes under different metabolic conditions or treatments (e.g., C16:0 treatment has been shown to significantly increase ACSL5 expression) .

What are optimal quantification methods for ACSL5 expression in different experimental systems?

For robust quantification of ACSL5 expression, employ these methodological approaches tailored to specific experimental systems:

Western Blot Quantification:

• Capture digital images under non-saturating conditions.

• Use ImageJ or similar software for densitometric analysis .

• Normalize ACSL5 band intensity to loading controls (β-actin is commonly used) .

• Calculate relative expression using the formula: (ACSL5 intensity / β-actin intensity).

• Perform at least three independent experiments for statistical validity.

• Apply appropriate statistical tests (e.g., t-test for comparing two conditions, ANOVA for multiple conditions).

Immunohistochemistry Quantification:

• Use ImageJ software for quantitative measurement of immunopositivity .

• Employ either:

  • H-score method: Intensity (0-3) × percentage of positive cells

  • Automated positive pixel count

  • Mean optical density measurements

• Analyze multiple fields per sample (minimum 5-10 random fields).

• Include blinded assessment to eliminate observer bias.

• Compare with expression of relevant markers (Ki-67, p53) for functional correlation .

Quantitative RT-PCR for mRNA Expression:

• Design specific primers for ACSL5:

  • Forward: 5′-GTCATCTGCTTCACCAGTGG-3′

  • Reverse: 5′-CGTCAGCCAGCAACCGAATATCC-3′

• Use β-actin as internal control:

  • Forward: 5′-AGCGAGCATCCCCCAAAGTT-3′

  • Reverse: 5′-GGGCACGAAGGCTCATCATT-3′

• Calculate relative expression using the 2^(-ΔΔCT) method after normalizing to the internal control .

• Perform multiple technical replicates (at least triplicate) for each biological sample.

• Include no-template and no-RT controls to detect contamination.

Cell-Based Assays:

• For fluorescence-based detection, use flow cytometry for quantitative single-cell analysis.

• Apply automated image analysis software for high-content screening of immunofluorescence images.

• Correlate ACSL5 expression with functional readouts (proliferation, apoptosis, migration) .

How can ACSL5 antibodies be used to investigate the link between lipid metabolism and cancer?

ACSL5 antibodies are instrumental in elucidating the intricate relationship between lipid metabolism and cancer through these specialized methodological approaches:

• Treatment response studies:

  • Use ACSL5 antibodies to monitor expression changes following treatment with fatty acids, particularly palmitic acid (C16:0), which has demonstrated anti-tumor effects accompanied by ACSL5 upregulation in non-small cell lung cancer .

  • Research has shown that C16:0 concentrations from 50μM to 200μM for 48 hours induced significant ACSL5 upregulation (>20-fold at 200μM) compared to controls .

  • Employ immunoblotting to correlate ACSL5 expression levels with tumor suppression phenotypes.

• Signaling pathway integration:

  • Apply ACSL5 antibodies in conjunction with phospho-specific antibodies for ERK pathway components to map the signaling network.

  • Research has demonstrated that C16:0 treatment inhibits phosphorylated ERK protein expression in A549 cells while increasing ACSL5 levels, suggesting a mechanistic link .

  • Use inhibitor studies combined with ACSL5 immunodetection to establish causality in pathway relationships.

• In vivo tumor model analysis:

  • Utilize ACSL5 antibodies for immunohistochemical analysis of xenograft tumor tissues following treatment interventions.

  • Studies in nude mice with subcutaneously injected A549 cells showed C16:0 treatment significantly inhibited tumor growth while upregulating ACSL5 and p53 expression, and reducing Ki-67 levels .

  • Correlate ACSL5 expression patterns with tumor volume measurements and other molecular markers to establish functional relationships .

• Mechanistic intervention studies:

  • Apply ACSL5 antibodies to evaluate expression changes in functional studies involving ACSL5 knockdown.

  • Research has shown that siRNA-mediated ACSL5 knockdown reversed the anti-tumor effects of C16:0, resulting in increased malignant phenotypes (proliferation, migration, invasion) .

  • Use these approaches to establish ACSL5 as a potential therapeutic target in cancer.

• Metabolic profiling integration:

  • Combine ACSL5 immunodetection with metabolomic analysis to correlate protein expression with changes in cellular lipid profiles.

  • Investigate how altered ACSL5 expression affects fatty acid utilization, membrane lipid composition, and energy production in cancer cells.

What is the significance of ACSL5 subcellular localization and how can it be studied?

The subcellular localization of ACSL5 is critical to its function and can be methodically investigated using specialized approaches:

• Multi-organelle localization significance:

  • ACSL5 has been identified in multiple cellular compartments, including mitochondria, endoplasmic reticulum, and cell membrane .

  • This distribution suggests compartment-specific roles in lipid metabolism, with potential differential functions in each location.

  • Mitochondrial localization likely relates to fatty acid oxidation, while ER localization may be associated with membrane lipid synthesis and remodeling.

• Subcellular fractionation techniques:

  • Implement differential centrifugation to isolate mitochondrial, ER, and plasma membrane fractions.

  • Apply ACSL5 antibodies in Western blot analysis of each fraction to quantify relative distribution.

  • Include organelle-specific markers as controls (e.g., VDAC for mitochondria, calnexin for ER, Na⁺/K⁺-ATPase for plasma membrane).

• Confocal microscopy co-localization studies:

  • Utilize immunofluorescence with ACSL5 antibodies together with organelle-specific markers.

  • Apply super-resolution microscopy techniques for precise localization.

  • Implement Manders' or Pearson's correlation coefficients to quantify co-localization.

  • Analyze dynamic localization changes under different metabolic conditions or treatments (e.g., fatty acid loading, starvation, C16:0 treatment) .

• Proximity ligation assays:

  • Employ this technique to detect ACSL5 interactions with organelle-specific proteins.

  • This approach can reveal function-specific protein complexes in different subcellular compartments.

• Functional impact of localization:

  • Design experiments using localization mutants (adding or removing targeting sequences) to direct ACSL5 to specific organelles.

  • Apply ACSL5 antibodies to confirm altered localization.

  • Correlate localization changes with functional outcomes in lipid metabolism and cancer-related phenotypes .

How can ACSL5 antibodies be integrated with emerging single-cell technologies?

Integration of ACSL5 antibodies with cutting-edge single-cell technologies offers powerful new research capabilities:

• Single-cell proteomics applications:

  • Incorporate ACSL5 antibodies into mass cytometry (CyTOF) panels to simultaneously detect ACSL5 alongside dozens of other proteins at the single-cell level.

  • Develop conjugated ACSL5 antibodies compatible with single-cell Western blot technologies (e.g., ProteinSimple Milo).

  • Apply computational analysis to identify cell subpopulations with distinct ACSL5 expression profiles.

• Spatial transcriptomics integration:

  • Combine ACSL5 antibody-based immunohistochemistry with spatial transcriptomics to correlate protein expression with transcriptional profiles in tissue context.

  • This approach enables identification of niche-specific regulation of ACSL5 in complex tissues.

  • Particularly valuable for analyzing tumor microenvironments and metabolic zonation in tissues like liver.

• Live-cell imaging applications:

  • Develop cell-permeable ACSL5 antibody fragments or nanobodies for real-time tracking of endogenous ACSL5 in living cells.

  • Monitor dynamic changes in ACSL5 localization in response to metabolic challenges or cancer therapeutics.

  • Combine with lipid probes to simultaneously visualize ACSL5 activity and lipid metabolism.

• Microfluidic-based single-cell analysis:

  • Incorporate ACSL5 antibodies into microfluidic platforms for analyzing protein expression in circulating tumor cells or rare cell populations.

  • Correlate ACSL5 expression with functional assays performed on the same individual cells.

  • This approach is particularly valuable for translational cancer research, potentially linking ACSL5 expression to treatment response or metastatic potential.

• CRISPR screening with antibody-based readouts:

  • Apply ACSL5 antibodies as readouts in pooled CRISPR screens to identify genes that regulate ACSL5 expression or subcellular localization.

  • Combine with phospho-ERK antibodies to map the regulatory network connecting ACSL5 to the ERK pathway in cancer cells .

  • This approach can identify novel therapeutic targets in the ACSL5 regulatory network.

How can ACSL5 antibodies contribute to multi-omics research approaches?

ACSL5 antibodies can be strategically integrated into multi-omics research frameworks through these methodological approaches:

• Proteogenomic integration:

  • Correlate ACSL5 protein expression (detected via antibodies) with genomic and transcriptomic data to identify regulatory mechanisms.

  • Investigate whether genetic variants in the ACSL5 gene correlate with protein expression levels in different tissues or disease states.

  • Combine RNA-seq data with ACSL5 protein quantification to identify post-transcriptional regulations.

• Metabolomics correlation:

  • Use ACSL5 antibodies to stratify samples based on protein expression, then perform untargeted metabolomics.

  • Correlate ACSL5 levels with specific lipid metabolites to establish functional consequences of expression variations.

  • This approach has been valuable in understanding how ACSL5 upregulation following C16:0 treatment affects cellular metabolism in cancer cells .

• Spatial multi-omics:

  • Apply ACSL5 immunohistochemistry on serial tissue sections alongside spatial transcriptomics and metabolomics.

  • This integrated approach reveals tissue microenvironments where ACSL5-mediated metabolism influences disease progression.

  • Particularly relevant for cancer research, where metabolic compartmentalization contributes to tumor heterogeneity .

• Temporal multi-omics:

  • Monitor ACSL5 protein dynamics (using antibodies) alongside transcriptional, epigenetic, and metabolic changes during:

    • Disease progression models

    • Treatment response (e.g., C16:0 administration)

    • Cellular differentiation processes

  • This approach captures the temporal sequence of molecular events involving ACSL5.

• Systems biology model construction:

  • Use quantitative ACSL5 protein data from antibody-based detection to parameterize computational models of lipid metabolism.

  • Integrate these models with signaling pathway models (e.g., ERK pathway) to predict cellular responses to metabolic perturbations .

  • Validate model predictions experimentally using ACSL5 antibodies as readouts.