ANTR3 Antibody

What is ANT3 and what cellular functions does it perform?

ANT3 is a mitochondrial protein that functions as an ADP:ATP antiporter, mediating the import of ADP into the mitochondrial matrix for ATP synthesis and the export of ATP to fuel cellular processes. It is localized to the mitochondria and features methylated post-translational modifications. ANT3 is also known by several aliases including AAC3, ANT, and ANT2, and is part of the solute carrier family 25 member 6 (SLC25A6) . The protein plays a critical role in cellular energy metabolism by facilitating the exchange of adenine nucleotides across the inner mitochondrial membrane, which is essential for oxidative phosphorylation and cellular respiration processes.

What applications are ANT3 antibodies commonly used for in research?

ANT3 antibodies are utilized for multiple research applications including Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), Immunofluorescence (IF), and Flow Cytometry . These antibodies enable researchers to detect, localize, and quantify ANT3 protein in various biological samples. Western blotting is particularly useful for determining ANT3 protein expression levels, while immunohistochemistry and immunofluorescence help visualize its distribution within tissues and cells. Flow cytometry applications allow for quantitative analysis of ANT3 in cell populations, providing insights into expression patterns across different cell types or under varying experimental conditions.

What species reactivity should be considered when selecting an ANT3 antibody?

When selecting an ANT3 antibody, researchers should consider the species reactivity based on their experimental model. Available ANT3 antibodies demonstrate reactivity with various species including Human (Hu), Mouse (Ms), and Rat (Rt) . Some antibodies are species-specific, while others display cross-reactivity across multiple species. For example, the antibody described in result is a rabbit polyclonal antibody that specifically reacts with human ANT3 for WB, IHC, and ELISA applications. When planning experiments, researchers should verify the documented reactivity of the antibody with their target species to ensure accurate and reliable results.

What is the recommended protocol for using ANT3 antibodies in Western Blotting?

For Western Blotting with ANT3 antibodies, researchers should follow these methodological steps:

• Sample Preparation: Extract total protein from your samples using appropriate lysis buffer containing protease inhibitors to prevent protein degradation.

• Protein Quantification: Determine protein concentration using Bradford or BCA assay.

• SDS-PAGE: Load 20-50 μg of protein per lane and separate by SDS-PAGE (10-12% gel recommended).

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane.

• Blocking: Block the membrane with 3-5% BSA or non-fat dry milk in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute ANT3 antibody (typically at 0.35 μg/mL or according to manufacturer's recommendation) in blocking buffer and incubate overnight at 4°C .

• Washing: Wash the membrane 3-5 times with TBST.

• Secondary Antibody: Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.

• Detection: Develop using ECL substrate and image using a chemiluminescence imaging system.

The expected molecular weight of ANT3 is approximately 33 kDa, which should be considered when analyzing Western blot results .

How can ANT3 antibodies be effectively used in immunofluorescence assays?

For effective immunofluorescence detection of ANT3:

• Cell Preparation: Culture cells on coverslips or use appropriate tissue sections.

• Fixation: Fix samples with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: Permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes (critical for accessing mitochondrial antigens).

• Blocking: Block with 1-5% BSA in PBS for 1 hour at room temperature.

• Primary Antibody: Apply diluted ANT3 antibody (approximately 10 μg/ml or as recommended) and incubate overnight at 4°C .

• Washing: Wash 3 times with PBS.

• Secondary Antibody: Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa-488) for 1 hour at room temperature.

• Counterstaining: Counterstain with DAPI for nuclei and consider mitochondrial markers like MitoTracker for colocalization studies.

• Mounting: Mount slides with anti-fade mounting medium.

For dual labeling experiments, consider co-staining with actin (using phalloidin) to examine potential cytoskeletal associations, as demonstrated in the C2C12 cell staining example .

What considerations are important when designing ELISA experiments with ANT3 antibodies?

When designing ELISA experiments with ANT3 antibodies, consider these methodological factors:

• Coating Buffer Selection: Use carbonate coating buffer (0.05 M Na₂CO₃, 0.035 M NaHCO₃, pH 9.6) for optimal antigen adsorption to the plate .

• Antigen Concentration: Coat plates with purified ANT3 protein at 1-5 μg/ml concentration overnight at 4°C.

• Blocking Optimization: Block with PBST containing 0.1% BSA for one hour at 37°C to reduce non-specific binding .

• Antibody Pre-incubation: For competitive ELISA formats, pre-incubate ANT3 antibody (0.35 μg/mL) with purified ANT3 protein before adding to coated wells.

• Dilution Series: Prepare log-fold dilutions of competing antigen starting from 1.0 mg/mL to generate reliable standard curves .

• Controls: Include both positive controls (known reactive samples) and negative controls (buffer only and irrelevant antibody controls).

• Detection System: Select appropriate enzyme-conjugated secondary antibodies and substrates based on sensitivity requirements.

• Data Analysis: Calculate affinity constants and binding parameters using appropriate mathematical models.

These considerations help ensure reproducible and reliable ELISA results when working with ANT3 antibodies.

How can computational modeling be integrated with experimental approaches to characterize ANT3 antibody binding?

Integrating computational modeling with experimental approaches for ANT3 antibody characterization involves:

• Antibody Variable Fragment Modeling: Create homology models of antibody variable fragments (Fv) using tools like MOE (Molecular Operating Environment) or online platforms such as PIGS server (http://circe.med.uniroma1.it/pigs)[3] .

• Target Protein Modeling: Generate a 3D structure model of ANT3 using PDB search homology modeling with the appropriate UniProt sequence (similar to the approach used for Ana o 3 modeling described in result ).

• Epitope Mapping Through Site-Directed Mutagenesis: Experimentally identify key residues in the antibody combining site through site-directed mutagenesis, then incorporate these constraints into computational models .

• Protein-Protein Docking: Use software like MOE Protein-Protein Dock application to predict interactions between the modeled antibody and ANT3 protein .

• Validation Through NMR Techniques: Apply saturation transfer difference NMR (STD-NMR) to define the antigen contact surface, which can be used to validate and refine computational models .

• Molecular Dynamics Simulations: Refine the 3D structure of the ANT3-antibody complex through molecular dynamics simulations to account for conformational flexibility .

• Selection of Optimal 3D Models: Use experimental metrics (binding affinity, epitope mapping data) to select the most probable 3D models from thousands of computationally generated options .

This integrated approach allows researchers to generate mechanistic insights into ANT3-antibody interactions that neither computational nor experimental methods alone could provide.

What factors influence ANT3 antibody specificity and cross-reactivity with related proteins?

Several factors influence ANT3 antibody specificity and potential cross-reactivity:

• Sequence Homology: ANT3 shares significant sequence similarity with other members of the adenine nucleotide translocase family, particularly ANT1, ANT2, and ANT4. Many commercially available antibodies recognize multiple ANT isoforms, as evidenced by antibodies described as "ANT1/ANT2/ANT3/ANT4" reactive .

• Epitope Characteristics:

  • Linear vs. Conformational Epitopes: Some antibodies recognize linear epitopes that remain intact after protein denaturation, while others target conformational epitopes that depend on the protein's tertiary structure .

  • Epitope Conservation: Highly conserved regions among ANT family members increase cross-reactivity potential.

• Antibody Generation Method: The immunization strategy used to produce the antibody influences specificity:

  • Recombinant Full-Length Protein: Antibodies generated against full-length ANT3 may have broader cross-reactivity .

  • Peptide-Specific Antibodies: Antibodies raised against unique peptide sequences from ANT3 may offer higher specificity.

• C-Terminal vs. N-Terminal Recognition: Some antibodies specifically target the C-terminal region of ANT proteins, as indicated by the "Anti-ANT1/ANT2/ANT3/ANT4 antibody (C-Term)" described in result .

• Post-translational Modifications: ANT3 features methylated post-translational modifications which may affect antibody recognition and specificity .

Researchers should carefully evaluate these factors when selecting antibodies for experiments requiring high specificity for ANT3 versus other ANT family members.

How can machine learning approaches improve ANT3 antibody design and specificity prediction?

Machine learning approaches can enhance ANT3 antibody design and specificity prediction through:

• Training on Experimental Selection Data: Machine learning models can be trained on phage-display experimental data to learn the relationship between antibody sequence and binding specificity, as demonstrated in result for other antibodies.

• Binding Energy Parameterization: Develop shallow neural networks to parameterize binding energy functions that capture the evolution of antibody populations across experiments .

• Mode Identification in Antibody Selection: Machine learning can identify distinct "modes" of selection that represent different binding behaviors, allowing for more nuanced understanding of antibody-antigen interactions .

• Sequence-to-Function Prediction: Models can predict the binding profiles of novel antibody sequences not present in training sets, facilitating the design of antibodies with customized specificity profiles against ANT3 .

• Eliminating Experimental Bias: Analysis of selection data at both amino acid and nucleotide levels using machine learning can confirm that selection occurs primarily through protein-ligand interactions rather than nucleotide-level biases or amplification artifacts .

• Conformational Epitope Prediction: Deep learning approaches similar to AlphaFold3 can help predict the docking of antibodies to ANT3, though current success rates for antibody docking (only 8.9% high-accuracy as noted in result ) indicate that multiple computational predictions should be experimentally validated.

These approaches allow for rational design of ANT3-specific antibodies with reduced cross-reactivity to other ANT family members.

How can researchers troubleshoot inconsistent Western blot results with ANT3 antibodies?

When troubleshooting inconsistent Western blot results with ANT3 antibodies, researchers should systematically address:

• Sample Preparation Issues:

  • Ensure complete mitochondrial protein extraction (critical for ANT3 detection)

  • Prevent protein degradation by using fresh protease inhibitors

  • Verify protein concentration using consistent quantification methods

• Antibody Selection and Handling:

  • Confirm antibody specificity for ANT3 vs. other ANT isoforms

  • Optimize antibody concentration (typically starting at 0.35 μg/mL)

  • Validate antibody lot-to-lot consistency with positive controls

• Protocol Optimization:

  • Adjust blocking conditions (3-5% BSA may be superior to milk for mitochondrial proteins)

  • Optimize incubation times and temperatures

  • Evaluate different membrane types (PVDF vs. nitrocellulose)

• Signal Detection Issues:

  • For weak signals: extend exposure time, increase antibody concentration, or use signal enhancement systems

  • For high background: increase washing steps, adjust blocking, or decrease antibody concentration

  • Consider using more sensitive detection methods for low abundance targets

• Expected Results Interpretation:

  • ANT3 should appear at approximately 33 kDa

  • Multiple bands may indicate cross-reactivity with other ANT family members

  • Post-translational modifications may cause slight shifts in molecular weight

• Positive and Negative Controls:

  • Include tissues/cells known to express ANT3 (e.g., erythrocytes)

  • Use ANT3 knockout/knockdown samples as negative controls when possible

By systematically addressing these factors, researchers can improve the consistency and reliability of Western blot results with ANT3 antibodies.

What approaches can resolve contradictory results between different antibody-based detection methods for ANT3?

When facing contradictory results between different antibody-based detection methods for ANT3, researchers should implement these systematic approaches:

• Comparative Method Validation:

  Detection Method	Strengths	Limitations	Validation Approach
  Western Blot	Confirms specific molecular weight, quantitative	Denatures proteins, may lose conformational epitopes	Validate with recombinant ANT3 standards
  Immunohistochemistry	Preserves tissue context, localization	Potential cross-reactivity, fixation artifacts	Compare with mRNA localization (ISH)
  Immunofluorescence	High-resolution subcellular localization	Autofluorescence interference	Co-stain with mitochondrial markers
  ELISA	Quantitative, high-throughput	Limited spatial information	Validate with competitive binding assays
  Flow Cytometry	Single-cell analysis, quantitative	Limited to cell suspensions	Include isotype and fluorescence minus one controls

• Epitope Accessibility Analysis: Different detection methods may access different epitopes based on protein folding, fixation, and sample preparation. Examine whether the antibody recognizes linear or conformational epitopes using reduced vs. non-reduced conditions .

• Multiple Antibody Validation: Use multiple antibodies targeting different epitopes of ANT3. For instance, compare results between antibodies that recognize C-terminal regions with those that target other domains .

• Orthogonal Detection Technologies: Supplement antibody-based methods with non-antibody approaches:

  • Mass spectrometry for protein identification

  • RNA expression analysis (qPCR, RNA-seq)

  • Functional assays measuring ADP/ATP transport

• Standardized Reporting Framework: Document all experimental variables systematically using the Minimum Information About Antibody Validation (MIAV) guidelines to enable proper comparison between methods.

• Cellular Context Consideration: Assess whether contradictory results stem from differences in cellular context (e.g., cell type, physiological state) rather than methodological issues.

This multi-faceted approach helps resolve contradictions and builds a more comprehensive understanding of ANT3 expression and function across different experimental contexts.

How should researchers interpret ANT3 antibody signals in the context of mitochondrial dynamics?

Interpreting ANT3 antibody signals in the context of mitochondrial dynamics requires consideration of several biological and technical factors:

• Subcellular Localization Patterns:

  • ANT3 is primarily localized to the mitochondrial inner membrane

  • Changes in ANT3 distribution patterns may indicate alterations in mitochondrial morphology, fission/fusion, or mitophagy

  • Co-staining with additional mitochondrial markers (e.g., TOMM20 for outer membrane, MitoTracker for matrix) helps distinguish genuine ANT3 signals from artifacts

• Expression Level Fluctuations:

  • Increased ANT3 expression may indicate enhanced mitochondrial biogenesis or compensatory upregulation

  • Decreased expression may suggest mitochondrial degradation or specific regulatory processes

  • Compare ANT3 levels with other mitochondrial proteins to distinguish specific ANT3 regulation from general mitochondrial changes

• Post-translational Modification Analysis:

  • ANT3 features methylated post-translational modifications that may affect antibody recognition

  • Changes in these modifications may appear as shifts in molecular weight or altered signal intensity

  • Consider using modification-specific antibodies when available to track specific ANT3 states

• Functional Correlation:

  • Correlate ANT3 antibody signals with functional measures of mitochondrial activity:

    • Oxygen consumption rate

    • Membrane potential measurements

    • ATP production assays

  • This correlation helps validate the biological significance of observed ANT3 expression patterns

• Temporal Dynamics Consideration:

  • Single time-point measurements may miss transient changes in ANT3 expression or localization

  • Time-course experiments provide more comprehensive understanding of ANT3 dynamics during mitochondrial remodeling processes

By integrating these considerations, researchers can more accurately interpret ANT3 antibody signals within the complex context of mitochondrial dynamics and cellular energetics.

How can ANT3 antibodies contribute to understanding the role of mitochondrial dysfunction in disease pathogenesis?

ANT3 antibodies offer powerful tools for investigating mitochondrial dysfunction in disease pathogenesis through several advanced approaches:

• Biomarker Development:

  • ANT3 expression patterns detected by specific antibodies may serve as biomarkers for mitochondrial dysfunction in various diseases

  • Quantitative analysis of ANT3 levels in patient samples could help stratify disease severity or progression

  • Longitudinal studies correlating ANT3 expression with clinical outcomes may reveal prognostic value

• Multiplexed Imaging Technologies:

  • Combining ANT3 antibodies with other mitochondrial markers in multiplexed imaging platforms (Imaging Mass Cytometry, CODEX, etc.)

  • This approach provides comprehensive spatial profiling of mitochondrial proteins in disease tissues

  • Analysis of co-expression patterns with other proteins may reveal disease-specific mitochondrial signatures

• Single-Cell Analysis:

  • ANT3 antibodies in flow cytometry and single-cell proteomic approaches enable analysis of mitochondrial heterogeneity at the single-cell level

  • This reveals how subpopulations of cells with distinct mitochondrial phenotypes contribute to disease progression

  • Integration with single-cell transcriptomics provides multi-omic insights into mitochondrial dysfunction

• Therapeutic Target Validation:

  • ANT3 antibodies can help validate the efficacy of therapeutics targeting mitochondrial dysfunction

  • Monitoring changes in ANT3 expression or localization following treatment provides mechanistic insights

  • In vitro and in vivo models treated with potential therapeutics can be assessed using ANT3 antibodies as readouts of mitochondrial health

• Functional Interaction Studies:

  • ANT3 antibodies in proximity ligation assays or co-immunoprecipitation studies reveal interactions with other proteins

  • Changes in these interaction networks may underlie disease mechanisms

  • Identification of novel ANT3-interacting partners opens new avenues for therapeutic intervention

These applications position ANT3 antibodies as valuable tools in understanding the mechanistic role of mitochondrial dysfunction across a spectrum of diseases, from neurodegenerative disorders to metabolic conditions and cancer.

What role might ANT3 antibodies play in investigating nuclear actin dynamics and DNA repair mechanisms?

ANT3 antibodies may contribute significantly to investigating the emerging connections between mitochondrial proteins, nuclear actin dynamics, and DNA repair through several innovative approaches:

• Nuclear-Mitochondrial Protein Translocation:

  • While ANT3 is primarily mitochondrial, some mitochondrial proteins can translocate to the nucleus under stress conditions

  • ANT3 antibodies can help track potential nuclear translocation events through subcellular fractionation and immunostaining

  • This approach may reveal previously unknown roles of ANT3 in nuclear processes

• Connection to Arp2/3 Complex and Nuclear Actin Polymerization:

  • The Arp2/3 complex (which includes Arp3) promotes nuclear actin polymerization that drives DNA repair mechanisms

  • While ANT3 and Arp3 are distinct proteins, investigating their potential functional relationships could reveal integration of mitochondrial and nuclear dynamics

  • ANT3 antibodies could be used alongside Arp3 antibodies in co-localization studies to explore these connections

• DNA Damage Response Investigations:

  • The Arp2/3 complex promotes homologous recombination (HR) repair by driving motility of double-strand breaks through nuclear actin polymerization

  • ANT3 antibodies could help investigate whether mitochondrial stress affects these nuclear repair processes

  • Dual labeling with ANT3 antibodies and DNA damage markers may reveal correlations between mitochondrial dysfunction and DNA repair efficiency

• Energy Supply for DNA Repair:

  • As an ADP/ATP transporter, ANT3 is critical for cellular energy distribution

  • ANT3 antibodies can help track changes in ANT3 expression or localization following DNA damage

  • This may reveal mechanisms by which cells ensure adequate ATP supply to energy-intensive DNA repair processes

• Integrated Stress Response Mechanisms:

  • Combining ANT3 antibodies with markers of integrated stress response pathways

  • May uncover how mitochondrial energy metabolism interfaces with nuclear processes during cellular stress

  • Could reveal novel therapeutic targets at the nexus of mitochondrial function and genome stability

This research direction represents a cutting-edge intersection of mitochondrial biology, nuclear dynamics, and genome maintenance, where ANT3 antibodies serve as important tools for mechanistic discovery.

How can advanced imaging techniques enhance the utility of ANT3 antibodies in research?

Advanced imaging techniques significantly enhance the research utility of ANT3 antibodies by providing unprecedented spatial, temporal, and quantitative insights:

• Super-Resolution Microscopy Applications:

  • STED (Stimulated Emission Depletion) microscopy: Resolves individual mitochondrial cristae structures (20-30 nm resolution) where ANT3 is localized

  • STORM/PALM techniques: Enable single-molecule localization of ANT3 within mitochondrial membranes

  • SIM (Structured Illumination Microscopy): Provides enhanced resolution for studying ANT3 distribution across larger mitochondrial networks

  • Implementation strategy: Optimize primary and secondary antibody concentrations to achieve high signal-to-noise ratio required for super-resolution techniques

• Live-Cell Imaging Innovations:

  • ANT3 antibody fragments (Fab, nanobodies) conjugated to cell-permeable fluorophores for real-time tracking

  • SNAP/CLIP-tag fusions with ANT3 for pulse-chase experiments to study protein turnover dynamics

  • FRET-based sensors incorporating ANT3 antibodies to detect conformational changes during transport activity

  • Methodological consideration: Validate that labeled antibodies or tag systems do not interfere with native ANT3 function

• Correlative Light and Electron Microscopy (CLEM):

  • ANT3 antibodies with dual fluorescent and electron-dense labels enable precise localization at both optical and ultrastructural levels

  • Gold-conjugated secondary antibodies for immunoelectron microscopy provide nanometer-scale localization of ANT3

  • Integration with 3D electron tomography reveals ANT3 distribution across the complex architecture of mitochondrial cristae

  • Technical requirement: Use specialized fixation protocols compatible with both immunofluorescence and electron microscopy

• Volumetric and Tissue Clearing Technologies:

  • Tissue clearing methods (CLARITY, iDISCO, CUBIC) combined with ANT3 antibodies for whole-organ imaging

  • Light-sheet microscopy enables rapid 3D imaging of ANT3 distribution throughout entire tissues

  • Serial section array tomography with ANT3 antibodies for high-resolution 3D reconstructions

  • Optimization need: Adjust antibody concentration and incubation times for deeper tissue penetration in cleared samples

• Quantitative Image Analysis Platforms:

  • Machine learning algorithms for automated segmentation of ANT3-positive structures

  • Spatial statistics to quantify co-localization with other mitochondrial markers

  • Time-series analysis for tracking dynamic changes in ANT3 distribution

  • Implementation challenge: Develop standardized image acquisition parameters to ensure reproducible quantification across experiments

These advanced imaging approaches transform ANT3 antibodies from simple detection tools into sophisticated probes for exploring the dynamic behavior and functional significance of ANT3 in diverse biological contexts.