ATPD Antibody

What is ATPD and why are antibodies against it important in research?

ATPD (ATP5D/ATP5F1D) is a crucial component of the mitochondrial membrane ATP synthase (F1F0 ATP synthase or Complex V), which produces ATP from ADP in the presence of a proton gradient across the mitochondrial membrane. The protein is part of the F1 domain and central stalk, which are essential for the rotary mechanism that couples ATP turnover to proton translocation . Antibodies against ATPD are vital research tools for studying mitochondrial function, energy metabolism disorders, and neurodegenerative diseases where mitochondrial dysfunction plays a role.

What sample types can be used with ATPD antibodies?

ATPD antibodies have demonstrated compatibility with multiple sample types in research applications. Commercial polyclonal ATPD antibodies have been validated for Mouse and Human samples . When working with these antibodies, researchers should consider the following sample preparations:

• Whole cell lysates (e.g., HepG2, MOLT4, Raji cell lines)

• Tissue lysates (e.g., brain tissue)

• Paraffin-embedded tissue sections (e.g., breast cancer tissue)

How can I validate the specificity of an ATPD antibody?

Validation of ATPD antibody specificity is crucial for reliable experimental results. Methodological approaches include:

• Western blot analysis using positive control samples (e.g., HepG2, MOLT4, or Raji cell lysates) to confirm the predicted band size of approximately 17 kDa for human ATPD

• Peptide neutralization/competition assays to confirm binding specificity

• Comparison of immunoreactivity between wild-type and ATPD-knockdown/knockout samples

• Immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein

• Analysis of antibody cross-reactivity with other ATP synthase subunits or related proteins

What are the optimal conditions for using ATPD antibodies in Western blotting?

For optimal Western blot results with ATPD antibodies, researchers should consider the following methodology:

• Sample preparation: Use standard SDS-PAGE (12%) for optimal resolution of the relatively small ATPD protein (17 kDa)

• Loading: 30-50 μg of total protein per lane is typically sufficient for detection

• Transfer: Use PVDF or nitrocellulose membranes with high transfer efficiency for small proteins

• Blocking: 5% non-fat dry milk or BSA in TBST is generally suitable

• Primary antibody dilution: Published research indicates optimal dilutions between 1:500 and 1:1000

• Secondary antibody selection: Anti-rabbit IgG conjugated to HRP works effectively with rabbit polyclonal ATPD antibodies

• Detection: Both chemiluminescence and fluorescence detection methods are compatible

Membrane Ponceau staining prior to antibody incubation can help verify successful protein transfer .

How can I optimize ATPD antibody use in immunohistochemistry?

For effective immunohistochemical detection of ATPD in paraffin-embedded tissues, the following methodology is recommended:

• Tissue fixation: Standard formalin fixation followed by paraffin embedding is compatible

• Section thickness: 4-6 μm sections are typically optimal

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

• Antibody dilution: A 1:250 dilution has been validated for human breast cancer tissue

• Detection system: Standard ABC (avidin-biotin complex) or polymer-based detection systems

• Counterstaining: Hematoxylin provides good nuclear contrast

• Controls: Include both positive control tissues (with known ATPD expression) and negative controls (primary antibody omitted)

What are the challenges in detecting ATPD in mitochondrial preparations?

Detection of ATPD in isolated mitochondrial fractions presents several methodological challenges:

• Maintaining structural integrity during isolation: Gentle isolation protocols are necessary to preserve the F1F0 ATP synthase complex structure

• Cross-contamination with other cellular compartments: Rigorous purification protocols are essential for accurate localization studies

• Distinguishing ATPD from other ATP synthase subunits: High-specificity antibodies and appropriate controls are crucial

• Detergent selection: The choice of detergent for mitochondrial solubilization affects epitope accessibility

For effective mitochondrial isolation, researchers should follow established protocols that include density gradient centrifugation and assess fraction purity using markers for different cellular compartments .

How can ATPD antibodies be used to study mitochondrial dysfunction in disease models?

ATPD antibodies provide valuable tools for investigating mitochondrial dysfunction in various disease models:

• Quantitative Western blotting to assess ATPD protein levels in disease versus control samples

• Immunohistochemistry to evaluate ATPD distribution and expression patterns in tissue sections

• Co-immunoprecipitation to study protein-protein interactions involving ATPD

• Proximity ligation assays to investigate in situ interactions between ATPD and other proteins

• Immunoelectron microscopy for subcellular localization studies

These approaches have proven valuable in studying conditions such as neurodegenerative diseases, cancer, and metabolic disorders where mitochondrial function is compromised.

What strategies can be employed to develop ATP-dependent antibodies for targeted therapeutics?

Advanced research is exploring the development of ATP-dependent antibodies for therapeutic applications, particularly in cancer treatment. These innovative approaches leverage the elevated extracellular ATP concentrations in tumor microenvironments:

• Phage display technology can be used to identify antibodies that bind to antigens only in the presence of ATP

• Crystallography analysis reveals that ATP can bind between the antibody-antigen interface, serving as a molecular switch for antigen binding

• In transgenic mouse models, ATP-switch antibodies have demonstrated tumor-specific binding with minimal binding to normal tissues, even when the antigen is expressed systemically

• This strategy allows therapeutic antibodies to overcome on-target off-tumor toxicity, a significant challenge in antibody therapeutics

How can ATPD antibodies contribute to studies of protein-protein interactions within the ATP synthase complex?

ATPD antibodies can facilitate the investigation of protein-protein interactions within the ATP synthase complex through several methodological approaches:

• Co-immunoprecipitation followed by mass spectrometry to identify interaction partners

• Proximity-dependent biotin identification (BioID) to map the protein interaction network surrounding ATPD

• Förster resonance energy transfer (FRET) microscopy to study dynamic interactions in living cells

• Blue native PAGE combined with Western blotting to analyze intact ATP synthase complexes and subcomplexes

• Cross-linking mass spectrometry (XL-MS) to map spatial relationships between ATPD and other complex components

These techniques provide insights into the structural organization and assembly of the ATP synthase complex, which is crucial for understanding its function in health and disease.

What are common issues when using ATPD antibodies and how can they be resolved?

Researchers may encounter several challenges when working with ATPD antibodies, including:

How do polyclonal and monoclonal ATPD antibodies compare in research applications?

The choice between polyclonal and monoclonal ATPD antibodies depends on specific research applications:

Polyclonal ATPD Antibodies:

• Recognize multiple epitopes on the target protein, enhancing detection sensitivity

• Useful for applications such as Western blotting and immunohistochemistry

• More tolerant of minor protein denaturation or conformational changes

• May show batch-to-batch variability requiring validation of each lot

• Generally less expensive and faster to produce

Monoclonal ATPD Antibodies:

• Target a single epitope, providing higher specificity

• Ideal for distinguishing between closely related protein family members

• Offer greater consistency between batches

• May be more sensitive to epitope masking or destruction

• Particularly valuable for quantitative applications requiring high reproducibility

What controls should be included when designing experiments with ATPD antibodies?

Proper experimental controls are essential for reliable interpretation of results when using ATPD antibodies:

• Positive controls: Include samples known to express ATPD (e.g., HepG2, MOLT4, Raji cell lysates, or brain tissue)

• Negative controls: Use samples from ATPD-knockout models or tissues known not to express the protein

• Technical controls:

  • Primary antibody omission control to assess secondary antibody specificity

  • Isotype control to evaluate non-specific binding

  • Peptide competition/neutralization assay to confirm antibody specificity

• Loading controls: Use established housekeeping proteins appropriate for the subcellular fraction being studied (e.g., VDAC or COX IV for mitochondrial fractions)

• Procedural controls: Include replicate samples to assess experimental variability

How are ATPD antibodies being used in innovative therapeutic research?

ATPD antibodies and related ATP synthase-targeting antibodies are finding novel applications in therapeutic research:

• Development of antibodies that exploit elevated extracellular ATP in tumor microenvironments for tumor-specific targeting

• Use of antibodies to monitor mitochondrial dysfunction in neurodegenerative diseases such as Alzheimer's disease

• Investigation of natural autoantibodies against mitochondrial proteins (including ATP synthase components) and their potential protective or pathogenic roles

• Application in vaccine development, where antibodies against ATP synthase components have shown protective efficacy against bacterial pathogens

What role do computational approaches play in ATPD antibody research and development?

Computational methods are increasingly important in antibody research and development, including for ATPD antibodies:

• The Assisted Design of Antibody and Protein Therapeutics (ADAPT) platform interleaves predictions and testing to guide affinity maturation of antibodies

• Computational approaches have successfully enhanced antibody binding affinity by an order of magnitude through point mutations

• In silico epitope prediction can identify antigenic regions of ATPD that are likely to elicit strong antibody responses

• Molecular dynamics simulations help predict antibody-antigen interactions and binding stability

• Structure-based computational approaches guide optimization of antibodies for therapeutic applications

These computational methods can significantly accelerate antibody development and reduce the need for extensive experimental screening.

How can ATPD antibodies contribute to understanding mitochondrial dynamics in live cells?

Advanced imaging techniques using ATPD antibodies or derived reagents are providing new insights into mitochondrial dynamics:

• Generation of Fab fragments or nanobodies against ATPD for live-cell imaging with minimal interference to normal mitochondrial function

• Development of ATPD-targeted fluorescent probes to monitor ATP synthase distribution and dynamics

• Application of super-resolution microscopy techniques (STED, PALM, STORM) to visualize ATPD localization at the nanoscale

• Implementation of FRET-based sensors incorporating ATPD-binding domains to detect conformational changes in the ATP synthase complex

• Correlation of ATPD distribution with mitochondrial membrane potential and ATP production using multiparameter imaging

These approaches allow researchers to investigate the relationship between ATP synthase organization, mitochondrial morphology, and cellular energetics in real-time.