ACOT7 Antibody

What is ACOT7 and why is it important in research?

ACOT7 belongs to the acyl coenzyme family and preferentially hydrolyzes palmitoyl-CoA, but has broad specificity for fatty acyl-CoAs with chain lengths of C8-C18. It plays crucial roles in fatty acid metabolism and has been implicated in multiple disease processes including hepatocellular carcinoma, Alzheimer's disease, and gastric cancer . ACOT7 is particularly abundant in brain tissue, suggesting a neuroprotective function by regulating neuronal fatty acid metabolism .

What types of ACOT7 antibodies are commercially available?

Several validated ACOT7 antibodies are available to researchers, as summarized in the table below:

These antibodies have been raised against different epitopes of ACOT7, with some targeting specific regions or isoforms, allowing researchers to select the most appropriate antibody for their specific application .

How should ACOT7 antibodies be stored and handled?

For optimal performance and longevity of ACOT7 antibodies:

• Store at -20°C for long-term storage (12 months stability)

• For short-term use (up to 1 month), storage at 4°C is acceptable

• Avoid repeated freeze-thaw cycles by making small aliquots before freezing

• Most ACOT7 antibodies are supplied in PBS with stabilizers such as 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations may contain 0.1% BSA for additional stability

• Allow antibodies to reach room temperature before opening the vial

• Briefly centrifuge before opening to collect all liquid at the bottom of the tube

What are the optimal Western blot conditions for ACOT7 detection?

For successful Western blot detection of ACOT7:

• Sample preparation: Prepare total homogenates in sucrose medium (10 mM Tris, 1 mM EDTA, 250 mM sucrose) or cell lysates in standard lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100)

• Loading and electrophoresis: Load 20-50 μg of protein on 8% or 12% SDS-PAGE gels

• Transfer: Transfer to PVDF membrane, block with 5% milk-TBST for 1 hour

• Antibody dilutions:

  • For polyclonal antibodies: 1:500-1:10000 (e.g., Proteintech 15972-1-AP at 1:7000)

  • For monoclonal antibodies: 1:500-1:50000 (e.g., Proteintech 68140-1-Ig)

• Expected molecular weight: 37-42 kDa (isoform dependent)

• Positive controls: Human, mouse, or rat brain tissue lysates (high endogenous expression)

• Secondary antibodies: HRP-conjugated anti-rabbit or anti-mouse IgG (typically 1:7000 dilution)

How can ACOT7 antibodies be utilized for immunohistochemistry?

For optimal immunohistochemical detection of ACOT7:

• Tissue preparation: Use formalin-fixed paraffin-embedded (FFPE) or frozen sections

• Antigen retrieval: Often necessary for FFPE sections (optimize based on specific antibody)

• Antibody dilutions: Start with manufacturer recommendations (e.g., 1:100 for Abcam ab85151)

• Expected staining pattern: Primarily cytoplasmic staining consistent with ACOT7's cellular localization

• Controls:

  • Positive control: Brain tissue (especially cerebral cortex)

  • Negative control: Omit primary antibody

• Visualization: Use appropriate detection system based on primary antibody species and isotype

• Counterstain: Hematoxylin provides good nuclear contrast for cytoplasmic ACOT7 staining

What protocols are effective for ACOT7 ELISA assays?

For quantitative ELISA detection of ACOT7:

• Equilibrate all reagents to room temperature (minimum 30 minutes)

• Use 96-well plates pre-coated with anti-ACOT7 antibody

• Add standards and samples, incubate for 2 hours at 37°C

• Remove liquid and add 100 μL of biotin-conjugated detection antibody, incubate for 1 hour at 37°C

• Wash three times with wash buffer

• Add avidin-conjugated HRP, incubate for 1 hour at 37°C

• Wash five times

• Add TMB substrate, incubate for 15-30 minutes at 37°C

• Add stop solution and read at 450 nm

• Interpolate sample values from standard curve using GraphPad Prism or similar software

This protocol has been successfully used to quantify ACOT7 in human serum for Alzheimer's disease biomarker studies .

Why might I observe multiple bands in ACOT7 Western blots?

Multiple bands in ACOT7 Western blots are common and may be attributed to:

• Multiple isoforms: ACOT7 has 7 known isoforms with molecular weights ranging from 27-40 kDa. Isoform 4 is expressed exclusively in brain tissue

• Post-translational modifications: Phosphorylation or other modifications can cause shifts in apparent molecular weight

• Proteolytic degradation: Add fresh protease inhibitors to all buffers and keep samples cold

• Cross-reactivity: ACOT7 antibodies may detect other ACOT family members due to sequence homology

• Non-specific binding: Optimize blocking conditions and antibody dilutions

Resolution strategies:

• Include recombinant ACOT7 protein as a positive control

• Use ACOT7 knockdown samples as negative controls

• Compare results with multiple antibodies targeting different epitopes

• Perform subcellular fractionation to confirm expected localization (cytosol/membrane)

How can I validate ACOT7 antibody specificity?

To ensure ACOT7 antibody specificity:

• Genetic validation:

  • Use ACOT7 knockdown or knockout samples

  • Validated siRNA sequences:

    • si1-ACOT7: 5′-AGACCGAGGACGAGAAGAADTDT-3′

    • si2-ACOT7: 5′-GUGCAGGUCAACGUGAUGUDTDT-3′

• Expression system validation:

  • Test in overexpression models (e.g., Flag-tagged ACOT7)

  • Compare staining patterns between wildtype and overexpression systems

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be reduced or eliminated if antibody is specific

• Multi-antibody approach:

  • Compare results using antibodies from different manufacturers

  • Look for consistent patterns with antibodies targeting different epitopes

What are common pitfalls when using ACOT7 antibodies for immunofluorescence?

Common challenges and solutions for ACOT7 immunofluorescence include:

• High background fluorescence:

  • Increase antibody dilution

  • Use more stringent blocking (5% BSA or 10% normal serum)

  • Include longer or additional washing steps

  • Use appropriate negative controls

• Weak or absent signal:

  • Optimize antigen retrieval for fixed samples

  • Decrease antibody dilution

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal amplification systems

• Non-specific nuclear staining:

  • Since ACOT7 is primarily cytoplasmic, nuclear staining may indicate non-specific binding

  • Pre-absorb antibody with nuclear extracts

  • Use alternative blocking reagents

• Auto-fluorescence:

  • Include auto-fluorescence quenching steps (e.g., Sudan Black B treatment)

  • Use narrow bandpass filters to reduce interference

How can ACOT7 antibodies be used to study transcriptional regulation?

ACOT7 antibodies can be employed in chromatin immunoprecipitation (ChIP) assays to study transcriptional regulation:

• Chromatin Immunoprecipitation (ChIP):

  • In HCC research, ChIP assays were conducted to investigate KLF13 binding to the ACOT7 promoter

  • Protocol: Flag-KLF13-overexpressing cells were analyzed using a ChIP enzymatic kit

  • qPCR primers: forward 5ʹ-GAAGGCAGCTAAGGCCCTG-3ʹ and reverse 5ʹ-GAGAGTCGTGGGCGGAAC-3ʹ

  • Controls: normal rabbit IgG and anti-Flag antibodies

• Reporter assays:

  • ACOT7 promoter constructs can be used to study transcriptional regulation

  • Combine with Western blot validation of transcription factor expression

• Expression correlation analyses:

  • ACOT7 antibodies can verify protein-level correlations with transcription factors identified through bioinformatic analyses

What role does ACOT7 play in cancer and how can antibodies help investigate this?

ACOT7 antibodies have revealed important roles in multiple cancer types:

ACOT7 antibodies enable these investigations through expression profiling, mechanism studies, and validation of genetic manipulations.

How is ACOT7 emerging as a biomarker for Alzheimer's disease?

Recent research has identified ACOT7 as a promising serum biomarker for Alzheimer's disease:

ACOT7 antibodies facilitated this discovery through:

• Western blot analysis showing 47% increase in AD patient serum

• ELISA-based quantification using anti-ACOT7 antibodies

• Cellular studies showing ACOT7's impact on AD-related proteins (BACE1, Aβ42, APP)

These findings suggest ACOT7 may be a superior serum biomarker compared to traditional Aβ42/40 ratio, with potential for clinical translation .

How might novel antibody technologies enhance ACOT7 research?

Emerging antibody technologies could significantly advance ACOT7 research:

• Single-domain antibodies (nanobodies):

  • Smaller size (15 kDa vs. 150 kDa for conventional antibodies)

  • Better tissue penetration for in vivo imaging

  • Potential for improved detection of ACOT7 in complex tissues

• Recombinant antibody fragments:

  • Defined production without batch-to-batch variation

  • Site-specific conjugation options for precise labeling

  • Engineered for improved stability and affinity

• Bispecific antibodies:

  • Simultaneously target ACOT7 and interaction partners

  • Study ACOT7 in specific cellular contexts or protein complexes

  • Potential therapeutic applications in diseases with ACOT7 dysregulation

• Intrabodies:

  • Engineered to function within living cells

  • Track ACOT7 localization and interactions in real-time

  • Monitor enzymatic activity rather than just expression

What opportunities exist for ACOT7 antibodies in single-cell and spatial biology?

Integration of ACOT7 antibodies with emerging single-cell technologies offers exciting research opportunities:

• Single-cell Western blotting:

  • Reveal heterogeneity in ACOT7 expression at single-cell resolution

  • Particularly valuable for cancer studies where expression may vary among subpopulations

• Mass cytometry (CyTOF):

  • Metal-tagged ACOT7 antibodies enable simultaneous detection with dozens of other proteins

  • Characterize ACOT7 expression in relation to cell lineage and activation markers

• Spatial proteomics:

  • Multiplex immunofluorescence with ACOT7 antibodies

  • Map expression patterns in tissue architecture

  • Correlate with neighboring cell types and tissue structures

• In situ sequencing combined with ACOT7 immunofluorescence:

  • Link ACOT7 protein expression with transcriptional profiles in tissue context

  • Understand regulatory relationships in situ

What neurological conditions beyond Alzheimer's disease might involve ACOT7?

Given ACOT7's high expression in brain and role in neuronal fatty acid metabolism, several neurological conditions warrant investigation:

• Epilepsy:

  • Decreased ACOT7 expression has been linked to mesial temporal lobe epilepsy

  • ACOT7 antibodies could validate this association in patient samples

  • Mouse models with conditional knockout in the nervous system (Acot7N−/−) show neurometabolic alterations

• Neurodevelopmental disorders:

  • ACOT7's role in brain development could be investigated through developmental expression studies

  • IHC analysis across developmental stages

• Neuroinflammatory conditions:

  • Given ACOT7's role in fatty acid metabolism, it may influence neuroinflammatory processes

  • Dual immunostaining with microglial markers could reveal relationships

• Other neurodegenerative diseases:

  • Based on ACOT7's promise as an Alzheimer's biomarker, investigation in Parkinson's disease, ALS, and other neurodegenerative conditions is warranted

  • Comparative expression studies using ACOT7 antibodies could identify disease-specific patterns

What validated siRNA sequences are available for ACOT7 knockdown studies?

The following siRNA sequences have been validated for ACOT7 knockdown:

Transfection protocols typically use Lipofectamine RNAiMAX (Invitrogen) following manufacturer's instructions. Knockdown efficiency should be validated by Western blot 48-72 hours post-transfection .

What transgenic models are available for ACOT7 research?

Several transgenic models have been developed for ACOT7 research:

• Conditional knockout models:

  • Nervous system-specific knockout (Acot7N−/−)

  • Exhibits alterations in brain fatty acid metabolism

  • Shows hypermetabolism, hepatic steatosis, and behavioral abnormalities

• Inducible overexpression models:

  • RIP2-rtTA transgenic mice with tetracycline-inducible Acot7 expression

  • Three lines (F15, F9, F26) showing different expression levels

  • Doxycycline administration induces ~14, 28, and 100-fold overexpression respectively

  • Expression restricted to insulin-positive β-cells

  • Useful for studying ACOT7's role in glucose metabolism

• Cell line models:

  • Stable ACOT7-overexpressing cell lines: SK-Hep1 and MHCC97H

  • Flag-tagged ACOT7 constructs for immunoprecipitation studies

  • Cytoplasmic (Acot7_cyt) and mitochondrial (Acot7_mit) isoform-specific constructs

These models provide valuable tools for investigating ACOT7 function in different physiological and pathological contexts.

What subcellular fractionation protocols work best for ACOT7 localization studies?

For effective subcellular fractionation to study ACOT7 localization:

• Homogenization buffer:

  • 10 mM Tris, 1 mM EDTA, 250 mM sucrose (pH 7.4)

  • Include protease inhibitor cocktail

• Cytosol/membrane separation:

  • Centrifuge total homogenates at 40,000 × g for 1 hour

  • Supernatant contains cytosolic fraction

  • Pellet contains membrane fraction

• Validation markers:

  • Cytosolic markers: GAPDH, β-tubulin

  • Membrane markers: Na+/K+ ATPase, calnexin

• Western blot detection:

  • Load equal amounts (20-50 μg) of each fraction

  • ACOT7 is primarily detected in cytosolic fraction

  • Isoform-specific antibodies may detect differential localization patterns

• Immunofluorescence confirmation:

  • Use organelle-specific markers for co-localization studies

  • Mitochondrial isoforms show punctate staining pattern