Acsl4 Antibody

What types of ACSL4 antibodies are available for research, and how do they differ?

ACSL4 antibodies are available as both monoclonal and polyclonal formats with various host species options:

• Monoclonal antibodies: Include mouse monoclonal options like ACSL4 Antibody (A-5) and ACSL4 Antibody (F-4), which offer consistent lot-to-lot reliability and specific epitope targeting

• Polyclonal antibodies: Including rabbit polyclonal options that recognize broader epitope ranges

• Recombinant antibodies: Newer options like Proteintech's 81196-1-RR that combine specificity with reproducibility

Each type offers different advantages depending on your experimental needs. Monoclonals provide high specificity for particular epitopes, while polyclonals recognize multiple epitopes, potentially enhancing signal detection in applications where the protein may be partially denatured.

How should I select between different ACSL4 antibody clones for my research?

Selection should be based on:

• Application compatibility: Verify validated applications (WB, IHC, IF, IP, ELISA) for your specific experimental needs

• Epitope recognition: Different clones recognize distinct protein regions - some target N-terminal sequences while others target internal or C-terminal regions

• Species cross-reactivity: Most commercial ACSL4 antibodies detect human, mouse, and rat ACSL4, but species reactivity should be verified for your model system

• Isoform detection: Consider whether the antibody detects specific splice variants (variant 1, variant 2, or both) if relevant to your research

• Published validation: Review literature citations where antibodies have been successfully employed in similar experimental contexts

What are the optimal protocols for using ACSL4 antibodies in Western blotting applications?

For Western blot detection of ACSL4:

• Sample preparation:

  • Use 30-100 μg of total cell lysates or tissue homogenates

  • Include protease inhibitors to prevent degradation

• Gel separation:

  • Use 7.5% SDS-PAGE gels for optimal resolution of the ACSL4 protein (74-79 kDa)

• Recommended dilutions:

  • For high-sensitivity detection: 1:5000-1:50000 for concentrated antibodies like Proteintech's 22401-1-AP

  • For standard applications: 1:100-1:500 for most commercial antibodies

• Visualization:

  • Both chemiluminescence (ECL Plus) and fluorescence detection methods are compatible

  • Normalize to β-actin or GAPDH for quantitative comparisons

• Expected band sizes:

  • Primary band at 79 kDa (full-length ACSL4)

  • Additional bands may appear at 74-75 kDa or 70 kDa representing different isoforms

How can I optimize ACSL4 detection in immunohistochemistry applications?

For optimal IHC results with ACSL4 antibodies:

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Antibody dilution range:

  • Most ACSL4 antibodies work optimally at 1:50-1:500 dilution for IHC

  • Titrate for each specific tissue type

• Positive control tissues:

  • Human liver tissue (high ACSL4 expression)

  • Rat brain tissue

  • Steroidogenic tissues (high endogenous expression)

• Detection systems:

  • Both HRP-based and fluorescent secondary detection systems are compatible

  • Multiple conjugated forms (HRP, PE, FITC, Alexa Fluor) are available for direct detection

• Cross-validation:

  • Consider using multiple antibody clones targeting different epitopes to confirm specificity

Why might I observe multiple bands or varying molecular weights when detecting ACSL4 by Western blot?

Multiple bands or varying molecular weights are common with ACSL4 detection and may represent:

• Splice variants: ACSL4 has two documented splice variants (variant 1: NM_004458 and variant 2: NM_022977)

• Protein isoforms: ACSL4 can exist as multiple isoforms:

  • 79 kDa (full-length protein)

  • 75 kDa (shorter isoform)

  • 70 kDa (possibly post-translationally modified form)

• Post-translational modifications: Evidence suggests ACSL4 can be ubiquitinated, which could alter migration patterns

• Sample preparation issues: Protein degradation can produce fragment bands

  • Consider adding proteasome inhibitors (e.g., MG132) when investigating ubiquitination

To confirm band specificity:

• Use positive control lysates (HepG2, rat liver tissue, HeLa cells)

• Perform genetic knockout validation experiments

• Compare results using antibodies targeting different ACSL4 epitopes

How can I confirm ACSL4 antibody specificity for my experimental system?

To validate ACSL4 antibody specificity:

• Genetic validation approaches:

  • ACSL4 knockdown (siRNA/shRNA)

  • CRISPR/Cas9-mediated knockout

  • Overexpression of tagged ACSL4 constructs

• Biochemical validation:

  • Immunoprecipitation followed by mass spectrometry

  • Pre-absorption with immunizing peptide when available

  • Parallel testing with multiple antibodies targeting different epitopes

• Controls for specific applications:

  • Western blot: Include lysates with known ACSL4 expression levels from HeLa, HepG2, or NIH/3T3 cells

  • IHC: Include tissues with documented high (liver, brain) and low ACSL4 expression

  • Immunofluorescence: Include secondary antibody-only controls to assess background

• Peptide competition assays:

  • When available, pre-incubate antibody with immunizing peptide

  • Should abolish specific signal while non-specific binding remains

How can ACSL4 antibodies be utilized to study ferroptosis mechanisms?

ACSL4 is a key regulator in ferroptosis, and antibodies can be used to:

• Monitor ACSL4 expression changes:

  • Western blot analysis of ACSL4 levels during ferroptosis induction

  • Immunofluorescence to assess subcellular localization changes during ferroptotic cell death

• Investigate ACSL4-dependent lipid metabolism:

  • Co-immunoprecipitation to identify ACSL4 protein interactions during ferroptosis

  • Analysis of ACSL4 expression correlation with lipid peroxidation markers

• Therapeutic targeting assessment:

  • Evaluate ACSL4 expression changes following treatment with ferroptosis inhibitors

  • Monitor ACSL4 levels in in vivo models using IHC

• Experimental design considerations:

  • ACSL4 converts free arachidonic acid (AA) into arachidonoyl-CoA, critical for generating lipid hydroperoxides during ferroptosis

  • ACSL4 expression changes have been documented in multiple ferroptosis-related pathologies, including cancer and kidney injury

  • Consider combining ACSL4 antibodies with lipid peroxidation assays for comprehensive ferroptosis assessment

What methodological approaches allow distinction between ACSL4 splice variants in experimental systems?

To differentiate between ACSL4 splice variants:

• RT-qPCR complementation:

  • Design primers to specifically detect variants 1 (NM_004458) or 2 (NM_022977)

  • Combine with antibody-based protein detection for comprehensive analysis

• Antibody selection strategies:

  • Select antibodies raised against unique regions of specific variants

  • Verify epitope mapping information from manufacturers

  • Some antibodies may recognize both variants, while others may be variant-specific

• Western blot pattern analysis:

  • Variant 2 is longer than variant 1, potentially producing different migration patterns

  • Compare observed band patterns with expected molecular weights

• Overexpression controls:

  • Generate expression constructs for individual variants with epitope tags

  • Use as positive controls for antibody specificity testing

How can ACSL4 antibodies be employed in multiparameter analyses of lipid metabolism pathways?

For comprehensive lipid metabolism studies:

• Multiplex immunofluorescence approaches:

  • Combine ACSL4 antibodies with antibodies against other lipid metabolism proteins (ACSL1, ACSL3, ACSL5)

  • Available conjugated formats include agarose, HRP, PE, FITC, and Alexa Fluor conjugates

• Co-immunoprecipitation strategies:

  • Use ACSL4 antibodies for IP followed by detection of interacting partners

  • Several antibodies have been validated for IP applications (0.5-4.0 μg for 1.0-3.0 mg total protein)

• Subcellular localization studies:

  • ACSL4 is predominantly localized in mitochondria, microsomes, and peroxisomes

  • Combine with organelle markers for co-localization analysis

• Functional correlation analyses:

  • Measure ACSL4 expression in parallel with assays for:

    • Arachidonic acid metabolism

    • Prostaglandin E2 production (ACSL4 modulates PGE2 secretion)

    • Cellular lipid content

What considerations are important when studying arachidonic acid-induced changes in ACSL4 expression?

When investigating arachidonic acid (AA) effects on ACSL4:

• Experimental design:

  • AA has been shown to selectively downregulate ACSL4 protein expression in hepatic cells

  • Consider dose-response relationships and time-course experiments

• Protein degradation mechanisms:

  • Evidence suggests ACSL4 may undergo ubiquitination and proteasomal degradation

  • Include proteasome inhibitors (e.g., MG132) to block degradation when studying these pathways

• Methodological approach:

  • Ubiquitination assays using co-transfection of HA-tagged ubiquitin and Flag-tagged ACSL4

  • Anti-HA or anti-Flag immunoprecipitation followed by Western blotting

  • Detection with anti-ACSL4 antibodies to assess modification patterns

• Control experiments:

  • Include empty vector transfection controls

  • Verify specificity with non-ubiquitinated ACSL4 standards

  • Consider parallel assessment of other ACSL family members to determine AA specificity

What quality control parameters should be considered when selecting ACSL4 antibodies?

When evaluating ACSL4 antibodies:

• Validation data:

  • Review Western blot images showing expected band sizes (79 kDa, 74-75 kDa)

  • Examine IHC/IF images for appropriate subcellular localization patterns

  • Check knockout/knockdown validation if available

• Immunogen information:

  • Some antibodies target N-terminal sequences (e.g., 14 AA peptide)

  • Others use recombinant fusion proteins (e.g., amino acids 1-280 of human ACSL4)

  • Epitope location may affect detection of modified or partially degraded protein

• Publication record:

  • Some antibodies have extensive citation records in peer-reviewed literature

  • For example, Proteintech's 22401-1-AP has been cited in 196 Western blot applications

• Storage and stability:

  • Most ACSL4 antibodies are stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Stable for one year after shipment when stored at -20°C