ATPAF2 Antibody

What is ATPAF2 and what is its biological significance?

ATPAF2, also known as ATP12 or ATP12p, functions as an essential assembly factor for the F1 component of mitochondrial ATP synthase. This protein specifically binds to the F1 alpha subunit, preventing the formation of nonproductive homooligomers during enzyme assembly . With a calculated molecular weight of 33 kDa, ATPAF2 is typically observed at approximately 30 kDa in Western blots, though a higher molecular weight band around 50 kDa may also be detected, potentially representing a dimer or complex form .

ATPAF2 plays a critical role in mitochondrial energy production, and its dysregulation has been implicated in various pathologies including metabolic disorders and neurodegenerative conditions . The protein's central function in ATP synthase assembly makes it an important target for studying mitochondrial biology and bioenergetics.

What applications have been validated for ATPAF2 antibodies?

Current commercially available ATPAF2 antibodies have been validated for multiple research applications. The following table summarizes the validated applications for several ATPAF2 antibodies:

When designing experiments, researchers should note that optimization of antibody dilution is recommended for each experimental system to obtain optimal results. The validation data indicates that these antibodies are suitable for detecting ATPAF2 in both human and mouse samples .

What positive controls are recommended for ATPAF2 antibody validation?

For Western blot applications, mouse liver tissue has been validated as a positive control for ATPAF2 detection . For immunohistochemistry applications, human stomach cancer tissue has been confirmed as a positive control . When establishing a new experimental system, these tissues can serve as appropriate positive controls to verify antibody performance.

The choice of positive control should align with the species being studied, with mouse liver tissue being particularly useful for rodent studies and human cancer tissues for human sample analysis. Proper antibody validation using these controls helps ensure the reliability and reproducibility of subsequent experimental results.

What are the optimal sample preparation methods for ATPAF2 detection in different applications?

For immunohistochemistry applications with ATPAF2 antibodies, antigen retrieval methodology significantly impacts detection quality. The recommended protocol involves using TE buffer at pH 9.0; alternatively, citrate buffer at pH 6.0 may be used . This optimization is critical when working with formalin-fixed, paraffin-embedded tissues.

For Western blot applications, standard protein extraction protocols are suitable for ATPAF2 detection. Given that ATPAF2 is a mitochondrial protein, mitochondrial isolation protocols may enhance detection sensitivity in samples with low expression levels. Sample preparation should include protease inhibitors to prevent degradation of the target protein during extraction.

When optimizing new protocols, it is advisable to test multiple antibody dilutions within the recommended ranges (WB: 1:500-1:3000, IHC: 1:50-1:500) to determine the optimal concentration for your specific sample type and experimental conditions .

How should researchers interpret multiple bands in ATPAF2 Western blots?

This larger band is not necessarily indicative of non-specific binding but may represent:

• Dimer formation

• Complex formation with other proteins

• Post-translational modifications

• Alternative splice variants

To verify band specificity, researchers should consider:

• Including appropriate positive and negative controls

• Performing peptide competition assays

• Using siRNA knockdown to confirm band identity

• Comparing results with multiple ATPAF2 antibodies targeting different epitopes

The presence of the 50 kDa band has been specifically noted in the literature and may correspond to dimer or complex forms of ATPAF2 , suggesting it represents biologically relevant information rather than experimental artifact.

How can ATPAF2 antibodies be utilized in studies of mitochondrial dysfunction and disease?

ATPAF2 antibodies provide valuable tools for investigating mitochondrial dysfunction in various pathologies. Given ATPAF2's role in ATP synthase assembly, alterations in its expression or localization may indicate compromised mitochondrial function.

For such studies, researchers should consider:

• Quantitative analysis of ATPAF2 expression levels using Western blot with appropriate loading controls

• Immunohistochemical evaluation of ATPAF2 distribution in disease tissue compared to healthy controls

• Co-localization studies with other mitochondrial proteins to assess potential changes in mitochondrial organization

• Correlation of ATPAF2 expression with functional measures of mitochondrial activity

ATPAF2 dysregulation has been implicated in metabolic disorders and neurodegenerative conditions . By studying ATPAF2 levels and function, researchers can gain valuable insights into the role of mitochondrial dysfunction in these pathologies and potentially identify new therapeutic targets.

What advanced techniques can be employed to study ATPAF2 protein interactions?

To investigate ATPAF2 interactions with other proteins, particularly the F1 alpha subunit of ATP synthase, several advanced techniques can be employed:

• Co-immunoprecipitation (Co-IP): Using ATPAF2 antibodies to pull down protein complexes, followed by Western blot analysis for potential binding partners.

• Proximity Ligation Assay (PLA): This technique allows for the visualization of protein interactions in situ with high sensitivity and specificity by combining antibody recognition with DNA amplification.

• FRET/BRET analysis: These techniques enable the study of protein-protein interactions in living cells by measuring energy transfer between fluorophores or bioluminescent molecules attached to potential interaction partners.

• Mass spectrometry-based approaches: After immunoprecipitation with ATPAF2 antibodies, mass spectrometry can identify novel interaction partners.

When designing these experiments, it's important to consider that ATPAF2 specifically binds to the F1 alpha subunit and may have additional uncharacterized interactions within the mitochondrial environment .

How can multiplexed detection systems be implemented for ATPAF2 analysis?

For comprehensive analysis of mitochondrial biology, simultaneous detection of ATPAF2 alongside other mitochondrial proteins can provide valuable contextual information. The monoclonal antibody (68351-2-PBS) has been validated for cytometric bead array applications, enabling multiplexed protein detection .

Multiplexed approaches for ATPAF2 analysis include:

• Multiplex flow cytometry: Using differentially labeled antibodies against ATPAF2 and other mitochondrial proteins.

• Cytometric bead array: The matched antibody pair (68351-1-PBS capture and 68351-2-PBS detection) has been specifically validated for this application .

• Multiplex immunofluorescence imaging: Using spectrally distinct fluorophores conjugated to antibodies against ATPAF2 and other targets.

• Sequential immunostaining: For tissue sections where multiple targets need to be visualized simultaneously.

These approaches allow researchers to analyze ATPAF2 in the context of other mitochondrial proteins, providing a more comprehensive understanding of mitochondrial structure and function in various experimental conditions.

How can ATPAF2 antibodies contribute to cancer research?

ATPAF2 antibodies have potential applications in cancer research, particularly in investigating mitochondrial dysfunction in tumor cells. The elevated extracellular ATP concentration in the tumor microenvironment represents a unique metabolic feature that could be exploited for targeted therapies .

Research applications include:

• Analysis of ATPAF2 expression in different cancer types: IHC applications using ATPAF2 antibodies have been validated in human stomach cancer tissue , suggesting utility in oncology research.

• Investigation of mitochondrial bioenergetics in cancer: Changes in ATPAF2 expression or localization may correlate with alterations in energy metabolism characteristic of cancer cells.

• Development of ATP-dependent targeting strategies: Recent research has demonstrated the potential to exploit elevated extracellular ATP in the tumor microenvironment for targeted therapeutic approaches . ATPAF2 antibodies could help elucidate the relationship between ATP synthase assembly and extracellular ATP levels.

• Study of mitochondrial dynamics in cancer progression: ATPAF2 antibodies can be used to monitor changes in mitochondrial function during cancer development and metastasis.

What novel approaches utilize ATP-dependent antibody targeting systems?

Recent advances in antibody engineering have demonstrated methods to exploit elevated extracellular ATP concentrations for targeted binding. Using phage display technology, researchers have developed 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.

This innovative approach shows particular promise for tumor-targeted therapies, as the tumor microenvironment typically contains elevated extracellular ATP levels compared to normal tissues. In a transgenic mouse model, ATP-switch antibodies demonstrated preferential binding to antigens in tumors with minimal binding in normal tissues and plasma, resulting in inhibited tumor growth .

While not directly related to ATPAF2 antibodies, these findings suggest potential applications for developing ATP-dependent ATPAF2 targeting strategies that could enhance specificity for pathological tissues with dysregulated ATP metabolism.

How should researchers address common challenges in ATPAF2 antibody applications?

When working with ATPAF2 antibodies, researchers may encounter several technical challenges. Here are methodological approaches to address common issues:

• Non-specific binding in Western blots:

  • Increase blocking time or concentration

  • Optimize antibody dilution within recommended ranges (1:500-1:3000)

  • Include additional washing steps

  • Consider using different blocking agents (BSA vs. milk)

• Weak or absent signal in IHC:

  • Verify antigen retrieval method (recommended: TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Optimize antibody concentration within recommended range (1:50-1:500)

  • Extend primary antibody incubation time

  • Consider signal amplification systems

• Background issues in immunofluorescence:

  • Increase washing duration or frequency

  • Use appropriate blocking sera matching secondary antibody host

  • Include additional blocking steps for endogenous peroxidase or biotin

  • Titrate primary and secondary antibody concentrations

For all applications, it is recommended that the antibody be titrated in each testing system to obtain optimal results, as sensitivity may be sample-dependent .

What are the optimal storage conditions for maintaining ATPAF2 antibody activity?

To maintain optimal activity of ATPAF2 antibodies, proper storage conditions are essential. Based on manufacturer recommendations:

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

• Antibodies are typically stable for one year after shipment when stored properly .

• For antibodies in liquid form with glycerol, aliquoting is unnecessary for -20°C storage .

• Some preparations may contain 0.1% BSA and/or 0.02% sodium azide as preservatives .

Working aliquots can be maintained at 4°C for short periods (1-2 weeks), but should be returned to -20°C for long-term storage to prevent activity loss. Repeated freeze-thaw cycles should be avoided as they can lead to decreased antibody performance through denaturation and aggregation.