ACSF3 Antibody

What is ACSF3 and what cellular functions does it perform?

ACSF3 (acyl-CoA synthetase family member 3) is a member of the ATP-dependent AMP-binding enzyme protein family localized in the mitochondria. It has a reported length of 576 amino acid residues with a molecular mass of 64.1 kDa . Functionally, ACSF3 catalyzes the initial reaction in intramitochondrial fatty acid synthesis by specifically activating malonate and methylmalonate, but not acetate, into their respective CoA thioesters .

Recent studies have demonstrated that ACSF3 promotes malonate detoxification, enhances mitochondrial metabolic flux, and is required for malonylation of mitochondrial proteins . This post-translational lysine modification can affect metabolic enzyme activity and alter cellular metabolism. The ACSF3 gene has been associated with the disease Combined malonic and methylmalonic aciduria, highlighting its clinical significance .

What applications are ACSF3 antibodies commonly used for?

ACSF3 antibodies are utilized in several immunodetection applications to study the expression, localization, and function of ACSF3 protein. The most common applications include:

• Western Blot (WB): Widely used to detect ACSF3 protein expression levels in cell or tissue lysates, with the expected band appearing at approximately 64 kDa .

• Enzyme-Linked Immunosorbent Assay (ELISA): Used for quantitative detection of ACSF3 in various samples .

• Immunohistochemistry (IHC): Applied to visualize the distribution and localization of ACSF3 in tissue sections .

These applications are essential for studying ACSF3's role in normal physiology and pathological conditions, particularly in research focused on mitochondrial metabolism and fatty acid synthesis disorders .

What species reactivity is typically available for ACSF3 antibodies?

Commercial ACSF3 antibodies show cross-reactivity with multiple species, which is important for comparative studies across different model organisms. Based on the available information, ACSF3 antibodies typically react with:

• Human ACSF3

• Mouse ACSF3

• Rat ACSF3

Some antibodies offer broader cross-reactivity, including:

• Bovine (Bv)

• Dog (Dg)

• Guinea Pig (GP)

• Horse (Hr)

• Yeast (Ys)

• Zebrafish (Zf)

When selecting an ACSF3 antibody for your research, it's important to verify the species reactivity in the manufacturer's documentation to ensure compatibility with your experimental model .

How should ACSF3 antibodies be stored and handled to maintain optimal activity?

For optimal preservation of ACSF3 antibody activity, the following storage and handling guidelines should be observed:

• Store antibodies at -20°C for long-term storage

• Avoid repeated freeze/thaw cycles as they can denature antibodies and reduce effectiveness

• Many commercial ACSF3 antibodies are provided in a buffer consisting of PBS with 0.01% thimerosal and 50% glycerol at pH 7.3, which helps maintain stability

• When working with the antibody, keep it on ice and return to storage promptly

• For working solutions, aliquot small volumes to minimize freeze/thaw cycles

• Follow manufacturer-specific recommendations for each antibody product, as formulations may vary

Proper storage and handling practices significantly extend antibody shelf-life and maintain consistent experimental results across studies.

What are the recommended protocols for Western Blot detection of ACSF3?

For optimal Western Blot detection of ACSF3, researchers should follow these methodological guidelines:

Sample Preparation:

• For mitochondrial proteins like ACSF3, isolation of mitochondrial fractions may improve signal-to-noise ratio

• Some protocols suggest TCA precipitation of mitochondrial preparations before solubilization in sample buffer to concentrate the protein

SDS-PAGE and Transfer:

• Use 9-10% polyacrylamide gels for optimal resolution of the 64 kDa ACSF3 protein

• For quantitative analysis, transfer proteins overnight at low voltage (30V) in cold 4 mM 3-(cyclohexylamino) propane sulfonic acid (pH 11) buffer

Antibody Incubation and Detection:

• Use primary ACSF3 antibody at dilutions of 1:500 to 1:3000, depending on the specific antibody and application

• Recommended blocking: 3% nonfat dry milk in TBST

• Secondary antibody: HRP-conjugated anti-rabbit IgG at approximately 1:10,000 dilution

• Detection using ECL chemiluminescent substrate with exposure times of approximately 60 seconds

Following this protocol should yield specific detection of ACSF3 at the expected molecular weight of 64 kDa, with minimal background interference.

How can researchers validate the specificity of ACSF3 antibodies?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For ACSF3 antibodies, consider these validation approaches:

• Knockout/Knockdown Controls: Compare immunoblots between wild-type cells and ACSF3 knockout or knockdown cells. CRISPR/Cas9-mediated knockout cells have been successfully generated for ACSF3 and serve as excellent negative controls .

• Overexpression Controls: Analyze cells overexpressing ACSF3 (wild-type or tagged versions) alongside untransfected controls to confirm signal enhancement.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before application to the sample. A specific antibody will show reduced or eliminated signal.

• Multiple Antibody Approach: Use antibodies targeting different epitopes of ACSF3 to confirm consistent detection patterns.

• Mass Spectrometry Validation: Immunoprecipitate ACSF3 using the antibody and verify the pulled-down protein by mass spectrometry.

• Cross-Reactivity Testing: Test the antibody against related proteins (other ACSF family members) to ensure specificity within the protein family.

These validation steps collectively strengthen confidence in antibody specificity and experimental results.

What are the relationships between ACSF3 and human diseases?

ACSF3 has been implicated in several metabolic disorders, and ACSF3 antibodies are valuable tools for studying these disease associations:

• Combined Malonic and Methylmalonic Aciduria (CMMA):

  • ACSF3 mutations are the primary cause of this rare metabolic disorder

  • Characterized by elevated levels of malonic acid and methylmalonic acid in urine

  • Patients may present with developmental delay, seizures, and metabolic acidosis

  • Western blot analysis using ACSF3 antibodies can help determine protein levels in patient samples

• Non-Alcoholic Fatty Liver Disease (NAFLD):

  • Hepatic fatty acid metabolism disorder is a key pathogenic mechanism in NAFLD

  • ACSF3 is involved in regulating fatty acid metabolism

  • Research has identified lysine acetylation sites in ACSF3 related to the mitochondrial deacetylase sirtuin 3 (SIRT3)

  • The SIRT3/ACSF3 pathway has become a target for therapeutic interventions, with natural compounds like protocatechuic acid (PCA) showing protective effects

• Mitochondrial Dysfunction Disorders:

  • ACSF3 knockout cells show impaired mitochondrial metabolism

  • Subtle differences in inner mitochondrial membrane proteins involved in oxidative phosphorylation were observed in ACSF3 KO cells

  • ACSF3 antibodies help detect these changes in protein expression and post-translational modifications

Understanding these disease associations provides opportunities for developing diagnostic tools and therapeutic strategies targeting ACSF3-related pathways.

How does post-translational modification of ACSF3 affect antibody detection?

Post-translational modifications (PTMs) of ACSF3 present complex challenges for antibody detection that researchers should address methodically:

Acetylation Effects:
ACSF3 contains lysine acetylation sites related to the mitochondrial deacetylase sirtuin 3 (SIRT3) . This acetylation can potentially mask or alter epitopes recognized by certain antibodies. When studying ACSF3 acetylation:

• Use pan acetyl-lysine antibodies in conjunction with ACSF3 immunoprecipitation to assess acetylation status

• Compare detection patterns between SIRT3-knockout and wild-type samples to identify acetylation-dependent epitope recognition

• Consider using acetylation-specific ACSF3 antibodies if available, or site-specific antibodies that target regions containing potentially acetylated lysines

Isolation Technique Considerations:
Different protein extraction methods can preserve or disrupt PTMs. For optimal detection of modified ACSF3:

• Use phosphatase and deacetylase inhibitors during extraction if studying phosphorylation or acetylation

• Consider native versus denaturing conditions based on whether conformational or linear epitopes are targeted by the antibody

• For mitochondrial proteins like ACSF3, use mitochondrial isolation protocols that preserve the native state of PTMs

Experimental Validation:
To confirm PTM effects on antibody detection:

• Compare detection before and after treatment with deacetylases (like SIRT3) or kinases/phosphatases

• Use multiple antibodies targeting different epitopes to create a comprehensive detection profile

• Implement mass spectrometry analysis to precisely identify modification sites and correlate with antibody detection efficiency

What are the optimal conditions for ACSF3 immunoprecipitation in studies of protein-protein interactions?

Immunoprecipitation (IP) of ACSF3 requires careful optimization to preserve physiologically relevant protein-protein interactions. Based on research methodologies described in the literature, the following protocol elements are recommended:

Lysis Buffer Composition:

• Use a gentle lysis buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% NP-40 or 0.5% Triton X-100

• Include protease inhibitors (e.g., complete protease inhibitor cocktail)

• For studies involving acetylation, add deacetylase inhibitors (e.g., 1 μM trichostatin A, 5 mM nicotinamide)

• For phosphorylation studies, include phosphatase inhibitors (e.g., 1 mM Na3VO4, 10 mM NaF)

Antibody Selection and Binding:

• Choose ACSF3 antibodies validated for IP applications

• Use 2-5 μg of antibody per 500 μg of protein lysate

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Incubate antibody with lysate overnight at 4°C with gentle rotation

Washing and Elution:

• Perform 4-5 washes with lysis buffer containing reduced detergent (0.1-0.2%)

• For studying weak interactions, use more gentle washing conditions

• Elute proteins using either acidic conditions (0.1 M glycine, pH 2.5), SDS sample buffer, or competing peptides

Verification Strategies:

• Confirm successful IP by Western blotting a small portion of the immunoprecipitate for ACSF3

• Analyze co-immunoprecipitated proteins by mass spectrometry or Western blotting with antibodies against suspected interaction partners

• Include appropriate controls: IgG control, input sample, and when possible, ACSF3 knockout cell lysate

This methodology has been successfully applied to study interactions between ACSF3 and components of fatty acid metabolism pathways .

How do ACSF3 mutations affect antibody epitope recognition in clinical samples?

ACSF3 mutations can significantly impact antibody epitope recognition, which has important implications for both research and clinical diagnostics. Understanding these effects requires a systematic approach:

Epitope Mapping and Mutation Analysis:

When working with clinical samples containing ACSF3 mutations, researchers should consider:

• Location of mutations relative to antibody epitopes:

  • Mutations within or near antibody epitopes may reduce or eliminate binding

  • Point mutations may have minimal effect on distant epitopes, while large deletions or truncations can cause significant detection issues

• Structural alterations:

  • Even distant mutations can affect protein folding and three-dimensional structure

  • Conformational antibodies are particularly susceptible to reduced binding due to structural changes

Methodological Approaches for Mutant ACSF3 Detection:

To address epitope recognition challenges:

• Multi-antibody strategy: Use multiple antibodies targeting different regions of ACSF3

  • N-terminal antibodies: Useful for detecting truncating mutations in C-terminal regions

  • C-terminal antibodies: Can confirm full-length protein expression

  • Internal epitope antibodies: Target conserved functional domains less likely to harbor mutations

• Custom antibody development:

  • For recurrent mutations in clinical populations, consider developing mutation-specific antibodies

  • These can be valuable for distinguishing wild-type from mutant protein in heterozygous samples

• Recombinant expression studies:

  • Express wild-type and mutant versions of ACSF3 (like the V372M variant identified in genetic databases)

  • Test antibody recognition across these variants to establish detection limitations

The clinical relevance of this approach is particularly evident in Combined Malonic and Methylmalonic Aciduria patients, where various ACSF3 mutations have been identified .

What controls should be included when using ACSF3 antibodies in various applications?

Proper experimental controls are essential for ensuring the validity and reproducibility of results when working with ACSF3 antibodies. The following controls should be incorporated based on specific applications:

For Western Blotting:

• Positive Controls:

  • Cell lines known to express ACSF3 (e.g., HEK293T cells)

  • Recombinant ACSF3 protein as a size reference

  • Tissue samples with confirmed ACSF3 expression (e.g., liver)

• Negative Controls:

  • ACSF3 knockout cells generated through CRISPR/Cas9

  • Cell lines with very low or no ACSF3 expression

  • Primary antibody omission control

• Loading Controls:

  • For whole-cell lysates: β-actin or GAPDH

  • For mitochondrial fractions: VDAC or other mitochondrial markers

For Immunoprecipitation:

• Input Control: 5-10% of pre-IP lysate to confirm target protein presence

• IgG Control: Non-specific IgG from the same species as the ACSF3 antibody

• Beads-Only Control: To identify proteins that bind non-specifically to beads

• Reverse IP: IP with antibodies against suspected interaction partners, then blot for ACSF3

For Immunohistochemistry/Immunofluorescence:

• Positive Tissue Control: Tissues known to express ACSF3

• Negative Tissue Control: Tissues with minimal ACSF3 expression

• Peptide Competition: Pre-incubation of antibody with immunizing peptide

• Primary Antibody Omission: To assess secondary antibody specificity

• Isotype Control: Non-specific IgG from the same species

For Functional Studies:

• Wild-type vs. Mutant Comparison: Compare wild-type ACSF3 with mutant versions (e.g., R354A and R354L)

• Rescue Experiments: Reintroduction of ACSF3 into knockout cells to restore function

• Activity Controls: Include no-ATP or no-CoA controls when measuring enzymatic activity

Implementing these controls systematically will help validate findings and troubleshoot potential issues in ACSF3 antibody applications.

How can researchers distinguish between different ACSF family members when using antibodies?

Distinguishing between ACSF family members requires careful antibody selection and validation strategies due to potential sequence homology and structural similarities. The following methodological approach is recommended:

Sequence Analysis and Antibody Selection:

• Epitope Targeting:

  • Select antibodies targeting unique regions with minimal sequence homology to other ACSF family members

  • N-terminal and C-terminal regions often show greater sequence divergence than catalytic domains

  • Review epitope information provided by manufacturers to assess potential cross-reactivity

• Specificity Validation:

  • Perform BLAST analysis of the immunizing peptide sequence to identify potential cross-reactive proteins

  • Request information from manufacturers about cross-reactivity testing against other ACSF family members

Experimental Validation Strategies:

• Overexpression Systems:

  • Express individual ACSF family members in cell systems with low endogenous expression

  • Test antibody reactivity against each overexpressed protein

  • Create a specificity profile for each antibody

• Knockout/Knockdown Validation:

  • Use CRISPR/Cas9 to generate specific knockouts for each ACSF family member

  • Validate antibody specificity by confirming signal loss only in the targeted knockout

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with the antibody of interest

  • Analyze precipitated proteins by mass spectrometry to identify any co-precipitated ACSF family members

Distinguishing Features of ACSF3:

ACSF3 has several unique characteristics that can help distinguish it from other family members:

• Subcellular Localization: ACSF3 is specifically localized to mitochondria, while other ACSF family members may have different subcellular distributions

• Substrate Specificity: ACSF3 activates malonate and methylmalonate, but not acetate . This unique substrate specificity can be used in functional validation studies.

• Molecular Weight: The precise molecular weight of ACSF3 (64.1 kDa) can help distinguish it from other family members that may migrate differently on SDS-PAGE.

• Disease Association: ACSF3's unique association with Combined malonic and methylmalonic aciduria provides another context for validation in patient samples.

By implementing these strategies, researchers can confidently distinguish ACSF3 from other ACSF family members and ensure specific detection in their experimental systems.

What troubleshooting approaches should be considered when ACSF3 antibody shows unexpected results?

When encountering unexpected results with ACSF3 antibodies, a systematic troubleshooting approach can help identify and resolve issues. Below is a comprehensive troubleshooting guide for common problems:

Problem 1: No Signal or Weak Signal

Potential causes and solutions:

• Low protein expression

  • Verify ACSF3 expression in your sample by RT-PCR

  • Use positive control samples known to express ACSF3

  • Consider enriching mitochondrial fractions since ACSF3 is mitochondrial

• Inefficient protein transfer (for Western blot)

  • Optimize transfer conditions - for ACSF3 (64 kDa), overnight transfer at low voltage (30V) in cold buffer might improve results

  • Verify transfer efficiency with reversible protein stains

• Antibody issues

  • Try different antibody concentrations (1:500 to 1:3000 for WB)

  • Ensure antibody storage conditions are appropriate (-20°C, avoid freeze/thaw cycles)

  • Consider a different ACSF3 antibody targeting an alternative epitope

Problem 2: Multiple Bands or Unexpected Band Size

Potential causes and solutions:

• Post-translational modifications

  • ACSF3 has known acetylation sites , which might affect migration

  • Compare results with and without deacetylase inhibitors

  • Test for phosphorylation with phosphatase treatment

• Splice variants

  • ACSF3 has alternatively spliced transcript variants

  • Verify which isoform your antibody should detect

  • Compare with recombinant ACSF3 protein standard

• Protein degradation

  • Use fresh samples and add protease inhibitors during extraction

  • Reduce sample heating time before loading

Problem 3: High Background or Non-specific Binding

Potential causes and solutions:

• Blocking issues

  • Optimize blocking conditions (3% nonfat dry milk in TBST is recommended)

  • Try alternative blocking agents (BSA, commercial blockers)

  • Increase blocking time

• Antibody concentration

  • Dilute primary and/or secondary antibodies further

  • Reduce incubation time

• Cross-reactivity

  • Perform peptide competition assay to confirm specificity

  • Use ACSF3 knockout cells as negative controls

Problem 4: Inconsistent Results Between Experiments

Potential causes and solutions:

• Sample variability

  • Standardize sample preparation protocols

  • Quantify protein loading accurately

  • Consider cellular conditions that might affect ACSF3 expression or modification

• Antibody stability

  • Aliquot antibodies to avoid repeated freeze/thaw cycles

  • Check antibody expiration and storage conditions

• Protocol inconsistencies

  • Document detailed protocols and standardize across experiments

  • Control for variables like incubation times and temperatures

This systematic approach to troubleshooting should help researchers resolve most issues encountered when working with ACSF3 antibodies, ultimately leading to more consistent and reliable experimental results.

How is ACSF3 antibody-based research contributing to our understanding of metabolic diseases?

ACSF3 antibody-based research has significantly advanced our understanding of several metabolic diseases through various mechanistic investigations:

Combined Malonic and Methylmalonic Aciduria (CMMA):

ACSF3 antibodies have been instrumental in characterizing the molecular pathology of CMMA, a rare metabolic disorder caused by mutations in the ACSF3 gene . This research has:

• Enabled the detection of aberrant ACSF3 protein expression in patient samples

• Facilitated structure-function studies of disease-causing mutations

• Helped establish genotype-phenotype correlations by quantifying mutant protein levels

• Supported the development of potential therapies targeting ACSF3 function or expression

Non-Alcoholic Fatty Liver Disease (NAFLD):

Recent research using ACSF3 antibodies has uncovered a novel pathway connecting SIRT3, ACSF3, and fatty acid metabolism in NAFLD pathogenesis :

• Antibody-based studies revealed that ACSF3 contains lysine acetylation sites regulated by the mitochondrial deacetylase SIRT3

• This SIRT3-ACSF3 interaction was found to be dysregulated in NAFLD models

• Western blot analysis with ACSF3 antibodies demonstrated that natural compounds like protocatechuic acid (PCA) could modulate this pathway

• These findings have opened new therapeutic avenues for NAFLD treatment targeting the SIRT3/ACSF3 pathway

Mitochondrial Dysfunction Disorders:

ACSF3 antibodies have helped elucidate the role of malonyl-CoA in mitochondrial homeostasis:

• Studies in CRISPR/Cas9-engineered ACSF3 knockout cells revealed impaired mitochondrial metabolism

• Immunoblotting showed altered expression of oxidative phosphorylation components in ACSF3-deficient cells

• This research established ACSF3 as essential for mitochondrial protein malonylation, a post-translational modification affecting metabolic enzyme activity

Future Clinical Applications:

The translational potential of ACSF3 antibody research includes:

• Biomarker Development: ACSF3 protein levels or post-translational modifications may serve as biomarkers for metabolic disease progression

• Therapeutic Monitoring: Antibody-based assays could monitor the effectiveness of interventions targeting the ACSF3 pathway

• Personalized Medicine: Characterization of patient-specific ACSF3 mutations could guide individualized treatment approaches

This research collectively demonstrates how ACSF3 antibodies are advancing both the fundamental understanding of metabolic diseases and the development of potential therapeutic strategies.

What are the emerging techniques for studying ACSF3 protein interactions and modifications?

Research on ACSF3 is rapidly evolving with the implementation of cutting-edge techniques to study its interactions and modifications. The following methodologies represent the frontier of ACSF3 research:

Advanced Protein Interaction Mapping:

• Proximity Labeling Techniques:

  • BioID or TurboID fusion with ACSF3 to identify proximity partners in the mitochondrial environment

  • APEX2-based proximity labeling to capture transient interactions in living cells

  • These approaches are particularly valuable for studying ACSF3 in its native mitochondrial context

• Crosslinking Mass Spectrometry (XL-MS):

  • Chemical crosslinking of ACSF3 complexes followed by mass spectrometry analysis

  • Provides structural information about interaction interfaces

  • Can capture weak or transient interactions that might be lost in traditional co-IP approaches

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps conformational changes in ACSF3 upon binding to partners or substrates

  • Helps identify allosteric regulation mechanisms

  • Particularly useful for studying how post-translational modifications affect ACSF3 structure

Post-Translational Modification Analysis:

• Site-Specific Antibody Development:

  • Generation of antibodies specific to acetylated ACSF3 at SIRT3-regulated sites

  • Enables tracking of acetylation status in different physiological and pathological conditions

• Middle-Down and Top-Down Proteomics:

  • Analysis of larger ACSF3 protein fragments or intact protein

  • Preserves combination patterns of multiple PTMs

  • Provides a more comprehensive view of ACSF3 modification states

• PTM Crosstalk Analysis:

  • Simultaneous monitoring of multiple modifications (acetylation, phosphorylation, malonylation)

  • Investigation of how one modification influences others

  • Particularly relevant given ACSF3's role in generating malonyl-CoA for protein malonylation

Live-Cell Imaging and Single-Molecule Techniques:

• FRET-Based Biosensors:

  • Development of biosensors to monitor ACSF3 activity or conformational changes in living cells

  • Real-time visualization of ACSF3 dynamics in response to metabolic perturbations

• Super-Resolution Microscopy:

  • Nanoscale visualization of ACSF3 distribution within mitochondria

  • Colocalization with interaction partners at sub-diffraction resolution

  • Tracking of dynamic changes in ACSF3 localization under different conditions

• Single-Molecule Enzymology:

  • Direct observation of ACSF3 enzymatic activity at the single-molecule level

  • Characterization of kinetic heterogeneity and conformational dynamics

These emerging techniques, combined with traditional antibody-based approaches, are expected to significantly advance our understanding of ACSF3 biology and its role in health and disease.