ACSF2 Antibody

What is ACSF2 and what role does it play in cellular metabolism?

ACSF2 (acyl-CoA synthetase family member 2) is an enzyme that plays an essential role in fatty acid metabolism by activating fatty acids to their CoA derivatives. Unlike other acyl-CoA synthetases, ACSF2 is phylogenetically distinct from known families of short-chain, long-chain, very long-chain, and bubblegum ACSs. Functionally, ACSF2 preferentially activates medium-chain fatty acids, particularly saturated fatty acids containing 6 to 10 carbons .

Biochemical characterization has revealed that ACSF2 has a Km apparent value of 24.4 μM for C10:0 (decanoic acid) and a Vmax apparent of 385 nmol/20min/mg in COS cell protein . This enzyme contains a critical lysine residue near the C-terminus that is evolutionarily conserved and essential for its catalytic activity; mutation of this residue to alanine completely abolishes enzyme activity .

ACSF2 is particularly significant in neuronal cells, where knockdown studies have demonstrated its importance for neurite outgrowth in response to retinoic acid, suggesting a crucial role in neuronal development . The protein migrates to nodes and points of neurite-neurite contact along with the presynaptic marker synaptophysin during neuronal differentiation, indicating a potential role in synapse formation .

What applications can the ACSF2 antibody be used for in research?

ACSF2 antibodies have been validated for multiple research applications, allowing comprehensive investigation of this protein across various experimental platforms. The primary validated applications include:

When performing immunohistochemistry with ACSF2 antibody, it is recommended to use TE buffer pH 9.0 for antigen retrieval, although citrate buffer pH 6.0 can serve as an alternative . These applications collectively enable researchers to investigate ACSF2 expression patterns, subcellular localization, protein interactions, and functional roles across different experimental systems.

Which species does the ACSF2 antibody show reactivity with?

Commercial ACSF2 antibodies have been validated for reactivity with multiple species, providing researchers flexibility in their experimental models. The species reactivity profiles for available ACSF2 antibodies are:

This cross-species reactivity is particularly valuable for comparative studies and for translating findings between animal models and human systems. The conservation of ACSF2 across species enables researchers to utilize these antibodies in various experimental contexts, from rodent models to human cell lines and tissues .

When transitioning between species or when using the antibody with untested species, it is advisable to perform preliminary validation experiments to confirm reactivity and specificity. This typically involves positive and negative controls alongside appropriate blocking strategies to ensure that observed signals are specific to ACSF2 rather than non-specific binding .

What is the recommended dilution for ACSF2 antibody in different applications?

Optimal dilution of ACSF2 antibody varies significantly depending on the specific application, sample type, and detection method. Following manufacturer recommendations while also conducting preliminary titration experiments is crucial for achieving optimal signal-to-noise ratios. The recommended dilution ranges for different applications are:

For Rabbit Recombinant ACSF2 Antibody (84033-2-RR):

For Rabbit Polyclonal ACSF2 Antibody (16140-1-AP):

It is important to note that these ranges represent starting points, and the optimal dilution may be sample-dependent . For this reason, it is recommended that researchers titrate the antibody in their specific testing system to obtain optimal results. For challenging samples or applications, preliminary experiments using a dilution series can help identify the optimal concentration that provides specific signal with minimal background.

Where is ACSF2 localized within cells, and does this vary between cell types?

ACSF2 exhibits interesting differential subcellular localization patterns that vary considerably depending on cell type, suggesting context-specific functional roles. This variation in localization may reflect cell-type specific metabolic requirements or regulatory mechanisms.

In neuronal cell lines (Neuro2a and P19), endogenous ACSF2 predominantly localizes to the Golgi region . This localization is dependent on intact Golgi architecture, as treatment with nocodazole (which disrupts the Golgi) leads to dispersal of ACSF2 throughout the cell . This Golgi localization suggests a potential role in lipid modification or trafficking in neuronal cells.

In contrast, in MA-10, HepG2, and skin fibroblast cells, ACSF2 demonstrates a mitochondrial localization pattern . This differential localization may reflect cell-specific metabolic demands, with mitochondrial localization potentially supporting fatty acid oxidation processes in these metabolically active cell types.

During neuronal differentiation, ACSF2 exhibits dynamic relocalization patterns. When Neuro2a cells are induced to differentiate with retinoic acid, ACSF2 migrates to nodes and points of neurite-neurite contact, colocalizing with the presynaptic marker synaptophysin . This specific relocalization suggests a potential role for ACSF2 in synapse formation and neuronal development.

The different localization patterns observed across cell types highlight the importance of determining ACSF2 localization in specific experimental contexts, as its function may vary accordingly.

How can I design an ACSF2 knockdown experiment using RNA interference?

Designing effective ACSF2 knockdown experiments requires careful consideration of targeting sequences, delivery methods, and validation strategies. Based on published protocols, the following methodological approach is recommended for successful ACSF2 knockdown using RNA interference:

siRNA Design and Targeting:
For transient knockdown, siRNAs targeting the coding sequence of mouse ACSF2 have proven effective. A validated siRNA sequence targets bp 8-27 of the mouse ACSF2 coding sequence: 5'-AAGCGAGCCATGGCTGTCTATCCTGTCTC-3' (sense) and 5'-AAATAGACAGCCATGGCTCGCCCTGTCTC-3' (antisense) . This targeting region has demonstrated significant knockdown efficiency in neuronal cell models.

Transfection Protocol:
For siRNA delivery, commercial transfection reagents such as Ambion siPORT Amine have been successfully employed. The optimized protocol involves:

• Incubating cells with siRNA in serum-free media for ten hours

• Adding fresh media containing fetal bovine serum

• Allowing 48-72 hours for effective protein knockdown

Stable Knockdown Strategy:
For long-term studies, stable ACSF2 knockdown cell lines can be generated using shRNA. The process involves:

• Inserting an shRNA-generating sequence equivalent to the siRNA sequence into a vector such as pSilencer 4.1 CMV-Hygro

• Transfecting the plasmid into target cells (e.g., Neuro2a)

• Selecting clonal lines using appropriate antibiotic (e.g., 300 mg/ml Hygromycin)

• Allowing two weeks for antibiotic-resistant colonies to emerge

• Expanding resistant colonies and validating knockdown efficiency

Validation of Knockdown:
Knockdown efficiency should be validated using multiple approaches:

• Indirect immunofluorescence to visualize protein reduction

• Western blotting for quantitative assessment of protein levels

• qPCR for mRNA expression analysis (a 57% reduction in mRNA has been observed with effective shRNA constructs)

Always include appropriate controls, such as scrambled siRNA/shRNA sequences, to differentiate specific effects from non-specific responses to the transfection process. In published studies, ACSF2 knockdown in Neuro2a cells resulted in significantly blunted neurite outgrowth in response to retinoic acid, providing a functional readout to confirm successful knockdown .

What methods are available for validating the specificity of ACSF2 antibody in my experiments?

Establishing antibody specificity is crucial for interpreting ACSF2 experimental results with confidence. Multiple complementary validation approaches should be employed:

Genetic Validation Methods:

• Knockout/Knockdown Controls: Utilizing ACSF2 knockdown or knockout cells/tissues provides the most robust specificity control. Published protocols have demonstrated that shRNA knockdown of ACSF2 results in reduced signal detection by ACSF2 antibodies in immunofluorescence and Western blot applications, confirming specificity . A validated shRNA construct (TL500159A) has shown 57% knockdown of ACSF2 mRNA and corresponding reduction in protein detection .

• Overexpression Systems: Comparing signal in cells overexpressing ACSF2 versus control cells can confirm antibody specificity. Published methods include transfecting full-length ACSF2 cDNA into COS-1 cells and performing parallel antibody validation in transfected and non-transfected cells .

Biochemical Validation Approaches:

• Immunoprecipitation-Mass Spectrometry: Performing immunoprecipitation with the ACSF2 antibody followed by mass spectrometry analysis of the captured proteins can confirm that ACSF2 is indeed the primary target.

• Western Blot Molecular Weight Verification: The calculated molecular weight of ACSF2 is 68 kDa (615 amino acids) . Observing a single band at this molecular weight in Western blot supports antibody specificity.

• Peptide Competition Assay: Pre-incubating the antibody with excess immunizing peptide should abolish specific staining in Western blot, IHC, or IF applications.

Cross-Validation Strategies:

• Multiple Antibody Comparison: Using different antibodies targeting distinct epitopes of ACSF2 (such as comparing results between 84033-2-RR and 16140-1-AP) should yield consistent detection patterns .

• Cross-Application Validation: Comparing ACSF2 detection across multiple techniques (WB, IHC, IF) should show consistent expression patterns in the same samples. Published data demonstrate successful ACSF2 detection in mouse lung tissue by Western blot and in human colon cancer tissue by IHC, providing cross-application validation opportunities .

These complementary approaches collectively provide robust validation of antibody specificity. For the most comprehensive validation, researchers should employ at least one genetic approach alongside biochemical and cross-validation strategies.

How does ACSF2 function change during neuronal differentiation and development?

ACSF2 undergoes significant functional and localization changes during neuronal differentiation, suggesting a developmentally regulated role in neurite outgrowth and synapse formation. Several key aspects of ACSF2's role in neuronal development have been characterized:

Functional Requirement for Neurite Extension:
ACSF2-deficient Neuro2a cells (generated using stable shRNA knockdown) display significantly impaired neurite outgrowth in response to retinoic acid treatment . This indicates that ACSF2 is not merely correlated with neurite extension but is functionally required for this developmental process. The mechanism likely involves ACSF2's ability to activate medium-chain fatty acids, which may provide essential lipid substrates or signaling molecules needed for membrane expansion during neurite extension.

Expression Pattern Throughout Development:
Northern blot analysis has revealed that ACSF2 mRNA is expressed in prenatal and newborn mouse brain, with expression patterns that change during development . This temporal regulation further supports a developmental role for ACSF2 in the nervous system.

Methodological Approaches for Studying ACSF2 in Neuronal Development:
To investigate ACSF2's role in neuronal differentiation, several experimental approaches have proven effective:

• Retinoic Acid-Induced Differentiation Model: Treating Neuro2a cells with retinoic acid induces differentiation, allowing for the study of ACSF2's role in this process .

• Immunofluorescence Co-localization Studies: Co-staining for ACSF2 and synaptophysin enables visualization of ACSF2 relocalization during differentiation .

• Quantitative Neurite Outgrowth Assays: Comparing neurite length and branching between ACSF2-knockdown and control cells provides functional readouts of ACSF2's developmental role .

• Live-Cell Imaging: Tracking ACSF2 localization in real-time during differentiation can provide insights into the dynamics of its developmental functions.

These findings collectively suggest that ACSF2 plays a specialized role in neuronal differentiation and development, potentially through providing activated fatty acids necessary for membrane expansion or signaling during neurite outgrowth and synapse formation .

What controls should I use when performing immunohistochemistry with ACSF2 antibody?

Implementing appropriate controls is essential for generating reliable and interpretable immunohistochemistry (IHC) data with ACSF2 antibody. A comprehensive control strategy should include:

Antibody Specificity Controls:

• Positive Tissue Controls: Include tissues known to express ACSF2. Based on validated IHC data, mouse colon tissue is recommended for the rabbit recombinant antibody (84033-2-RR) , while human colon cancer tissue and human skin tissue are recommended for the polyclonal antibody (16140-1-AP) . These positive controls confirm that the staining protocol is working properly.

• Negative Tissue Controls: Include tissues with minimal or no ACSF2 expression, or alternatively, use ACSF2-knockdown or knockout tissues if available. Absence of staining in these samples helps confirm antibody specificity.

• Primary Antibody Omission: Process sections through the entire IHC protocol but omit the primary ACSF2 antibody to identify any non-specific binding from the secondary detection system.

• Isotype Control: Use a non-specific IgG from the same host species (rabbit IgG) at the same concentration as the ACSF2 antibody to identify non-specific binding due to the antibody isotype.

Technical Controls:

• Antigen Retrieval Optimization: For ACSF2 IHC, the recommended antigen retrieval buffer is TE buffer pH 9.0, although citrate buffer pH 6.0 may also be used as an alternative . Compare both methods to determine optimal retrieval conditions for your specific tissue.

• Titration Series: Test multiple dilutions of the ACSF2 antibody to determine optimal concentration. For IHC, recommended dilutions range from 1:250-1:1000 for the recombinant antibody and 1:200-1:800 for the polyclonal antibody .

• Incubation Time Controls: Vary primary antibody incubation times (e.g., 1 hour at room temperature vs. overnight at 4°C) to optimize signal-to-noise ratio.

Validation Controls:

• Multi-method Validation: Compare IHC results with other detection methods such as immunofluorescence or Western blot using the same antibody on matched samples.

• Peptide Competition: Pre-incubate the ACSF2 antibody with the immunizing peptide before IHC staining. Specific staining should be blocked or significantly reduced.

• Multiple Antibody Validation: If possible, compare staining patterns using different antibodies targeting different epitopes of ACSF2.

Interpretation Controls:

• Serial Section Analysis: Examine serial sections stained for ACSF2 and related markers to understand the context of expression patterns.

• Counterstaining: Use appropriate counterstains (e.g., hematoxylin) to provide cellular context for ACSF2 localization.

Following these comprehensive control strategies will ensure robust and reliable ACSF2 immunohistochemistry results while minimizing the risk of false-positive or false-negative interpretations.

How can I optimize protein extraction protocols to maximize ACSF2 detection in Western blots?

Optimizing protein extraction for ACSF2 Western blot detection requires careful consideration of the protein's biochemical properties and subcellular localization. The following comprehensive approach addresses key aspects of sample preparation for optimal ACSF2 detection:

Subcellular Localization-Based Extraction:
Since ACSF2 exhibits differential subcellular localization depending on cell type (Golgi in neuronal cells; mitochondria in MA-10, HepG2, and skin fibroblasts) , extraction protocols should be tailored accordingly:

• For Golgi-localized ACSF2 (neuronal cells):

  • Include detergents that effectively solubilize Golgi membranes, such as 1% Triton X-100 or 0.5% CHAPS

  • Consider using sucrose gradient centrifugation for enrichment of Golgi fractions prior to Western blot

• For Mitochondrial ACSF2 (MA-10, HepG2, fibroblasts):

  • Use mitochondrial isolation buffers containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, pH 7.4

  • Include digitonin (0.025-0.1%) for selective permeabilization of the outer membrane while preserving mitochondrial integrity

Buffer Composition Optimization:
The following buffer components have been shown to improve ACSF2 extraction and stability:

Protease and Phosphatase Inhibition:
ACSF2 may be susceptible to degradation during extraction. Include a comprehensive protease and phosphatase inhibitor cocktail:

• Complete protease inhibitor cocktail (Roche or equivalent)

• 1 mM PMSF (added fresh before use)

• 5 mM NaF (phosphatase inhibitor)

• 1 mM Na3VO4 (phosphatase inhibitor)

• 2 mM β-glycerophosphate (phosphatase inhibitor)

Extraction Conditions:

• Temperature: Perform all extraction steps at 4°C to minimize protein degradation

• Mechanical Disruption: For tissue samples, use a Dounce homogenizer (10-15 strokes with tight pestle)

• Sonication: Brief sonication (3 x 10 seconds, 30% amplitude) can improve protein extraction, particularly for membrane-associated proteins

• Incubation Time: Limit extraction time to 30 minutes to minimize degradation

Sample Loading and Electrophoresis:

• Heat Denaturation: 5 minutes at 95°C in Laemmli buffer containing 5% β-mercaptoethanol

• Protein Loading: 20-50 μg total protein per lane (validated for detection of ACSF2 in mouse lung tissue)

• Gel Percentage: 10% SDS-PAGE is optimal for resolving the 68 kDa ACSF2 protein

Western Blot Optimization:

• Transfer Conditions: Use wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody Dilution: Use 1:500-1:1000 dilution of ACSF2 antibody (16140-1-AP) for Western blot

• Incubation Time: Overnight at 4°C for primary antibody incubation

This comprehensive protocol addresses the unique challenges of ACSF2 detection in Western blot applications, accounting for its differential subcellular localization and biochemical properties.