ATP5O Antibody

What is ATP5O and what cellular functions does it perform?

ATP5O (ATP synthase subunit O) is a critical component of the mitochondrial ATP synthase complex, specifically part of the peripheral stalk linking the F1 catalytic domain and F0 membrane-spanning proton channel. The N-terminus of ATP5O directly interacts with the F1 subunit, while the C-terminus interacts with F0 and is crucial for the oligomycin sensitivity of the H+ channel . With a molecular weight of approximately 23 kDa, ATP5O plays an essential role in oxidative phosphorylation and ATP production. The protein is encoded by the ATP5O gene (GenBank Accession Number BC021233) and has been identified to participate in multiple biological processes including respiratory electron transport chain (GO:0022904), ATP catabolic processes (GO:0006200), and mitochondrial ATP synthesis coupled proton transport (GO:0042776) .

Recent research has revealed that ATP5O expression is elevated in cancer-associated fibroblasts compared to normal fibroblasts in oral squamous cell carcinoma, reflecting increased oxidative phosphorylation requirements for ATP generation in these cells . Additionally, ATP5O has been identified as a potential biomarker for prostate and gastric cancers .

What applications can ATP5O antibodies be used for in research settings?

ATP5O antibodies have been validated for multiple research applications:

For Western blotting, ATP5O antibodies detect a band of approximately 23-25 kDa in various cell lines including A549, HEK-293, HeLa, HepG2, and tissue samples from multiple organs . When performing immunohistochemistry, antigen retrieval is recommended using TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 .

What is the difference between monoclonal and polyclonal ATP5O antibodies?

The selection between monoclonal and polyclonal ATP5O antibodies depends on your specific research requirements:

Monoclonal ATP5O Antibodies:

• Available options include mouse monoclonal (clone OTI2E9) and rabbit monoclonal (E7F4U)

• Recognize a single epitope, providing high specificity

• Exhibit consistent lot-to-lot performance

• Optimal for specific detection applications requiring minimal background

• Example: Bio-Rad's PrecisionAb mouse anti-ATP5O (clone OTI2E9) recognizes amino acids 24-213 of human ATP5O

Polyclonal ATP5O Antibodies:

• Multiple options from various manufacturers (Proteintech, Affinity Biosciences, Novus Biologicals)

• Recognize multiple epitopes on the ATP5O protein

• Generally provide stronger signal amplification

• Better suited for detecting proteins with low expression levels

• Example: Proteintech's rabbit polyclonal (10994-1-AP) is generated against ATP5O fusion protein Ag1458

The choice between these antibody types should consider factors such as the sensitivity requirements, target protein abundance, and the specific application methodology being employed.

What species reactivity can be expected from commercially available ATP5O antibodies?

Commercially available ATP5O antibodies demonstrate varying species reactivity profiles:

When selecting an ATP5O antibody for cross-species applications, researchers should consider the degree of sequence homology between species. The high conservation of ATP5O across mammalian species explains the broad reactivity of many of these antibodies. For novel or less-studied species, preliminary validation experiments are advised to confirm reactivity .

How should researchers optimize ATP5O antibody dilution for detecting low expression levels?

Optimizing ATP5O antibody dilution is critical for detecting low expression levels while maintaining signal-to-noise ratio. Begin with the manufacturer's recommended dilution range (e.g., 1:500-1:3000 for Western blot) and perform a titration experiment:

• Initial Dilution Series Testing:

  • Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:3000)

  • Test using consistent sample loading and detection conditions

  • For extremely low expression, start with more concentrated antibody dilutions (1:200-1:500)

• Sample Enrichment Techniques:

  • For mitochondrial proteins like ATP5O, perform mitochondrial fractionation to enrich the target

  • Use 10-20 μg of mitochondrial fraction instead of whole cell lysate

  • Consider immunoprecipitation to concentrate the protein before Western blotting

• Signal Enhancement Strategies:

  • Increase sample loading (up to 50 μg for whole cell lysate)

  • For Western blot, extend exposure time while monitoring background

  • Use high-sensitivity chemiluminescent substrates

  • For immunohistochemistry, consider signal amplification systems like tyramide signal amplification

• Optimization Considerations:

  • Different tissue/cell types may require different optimal dilutions

  • Polyclonal antibodies (like Proteintech 10994-1-AP or Novus Biologicals) may provide better sensitivity for low expression detection

  • Monitor background levels carefully as more concentrated antibody solutions may increase non-specific binding

According to Proteintech's recommendations, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" and may be "Sample-dependent" .

What are the best antigen retrieval methods for ATP5O detection in FFPE tissue sections?

Effective antigen retrieval is crucial for ATP5O detection in formalin-fixed paraffin-embedded (FFPE) tissue sections. Based on available data, the following methods are recommended:

• Heat-Induced Epitope Retrieval (HIER):

  • Primary Recommendation: TE buffer at pH 9.0

    • Prepare: 10 mM Tris, 1 mM EDTA, pH 9.0

    • Heat in pressure cooker, microwave, or water bath (95-98°C for 15-20 minutes)

  • Alternative Method: Citrate buffer at pH 6.0

    • Prepare: 10 mM sodium citrate buffer, pH 6.0

    • Same heating parameters as above

• Protocol Optimization:

  • For human tissues (lung cancer, colon cancer, testis, heart), particularly good results have been observed with the TE buffer pH 9.0 method

  • Begin with manufacturer-recommended dilutions (e.g., 1:50-1:500) and adjust based on signal strength

  • Include positive control tissues known to express ATP5O (e.g., heart tissue, which is rich in mitochondria)

• Special Considerations:

  • ATP5O is a mitochondrial protein, so tissues with high mitochondrial content generally show stronger signal

  • Allow slides to cool slowly to room temperature after heating to prevent tissue detachment

  • For multiplex IHC involving ATP5O, the pH 9.0 method typically provides better antigen retrieval for multiple targets

Using appropriate antigen retrieval methods significantly impacts staining quality, as ATP5O antibodies have been successfully used to detect the protein in multiple human tissues including lung cancer, colon cancer, testis, and heart tissue .

How do post-translational modifications of ATP5O affect antibody recognition?

Post-translational modifications (PTMs) of ATP5O can significantly impact antibody recognition and experimental outcomes. Researchers should consider the following when designing experiments:

• Known PTMs of ATP5O:
  ATP5O is subject to several post-translational modifications that may affect epitope accessibility:

  Site	PTM Type	Source
  R29	Methylation	UniProt
  Y35	Phosphorylation	UniProt
  Y41	Phosphorylation	UniProt
  T43	Phosphorylation	UniProt
  Y46	Phosphorylation	UniProt

• Impact on Antibody Recognition:

  • Phosphorylation at tyrosine residues (Y35, Y41, Y46) may alter epitope conformation

  • If the antibody's epitope includes or is adjacent to these modification sites, recognition may be impaired or enhanced

  • For example, Cell Signaling's E7F4U rabbit mAb is produced using a synthetic peptide corresponding to residues near the carboxy terminus , which may be less affected by the listed N-terminal PTMs

• Experimental Considerations:

  • Use phosphatase treatment controls if phosphorylation is suspected to interfere with detection

  • Consider using multiple antibodies targeting different epitopes of ATP5O

  • Recent research indicates that acetylation of ATP5O may occur through direct binding of claudin-10 (CLDN10) to ATP5O in the outer mitochondrial membrane, which affects mitochondrial function and metastasis in clear cell renal cell carcinoma

• Mitochondrial Processing:

  • ATP5O is synthesized as a precursor with a mitochondrial targeting sequence that is cleaved upon import

  • Antibodies raised against the full-length protein versus the mature form may recognize different species

Understanding the relationship between PTMs and epitope recognition is particularly important when studying ATP5O in disease contexts, as modification patterns may change in pathological conditions like cancer, where ATP5O has been identified as a potential biomarker .

What are the best approaches to validate ATP5O antibody specificity?

Rigorous validation of ATP5O antibody specificity is essential for generating reliable research data. The following comprehensive validation approach is recommended:

• Positive and Negative Control Samples:

  • Positive Controls: Use cell lines with known ATP5O expression such as A549, HEK-293, HeLa, HepG2, Caco-2, and HCT116 cells

  • Tissue Controls: Mouse heart, liver, placenta, and colon tissues demonstrate strong ATP5O expression

  • Negative Controls: Consider using ATP5O knockdown/knockout cells or siRNA-treated samples

• Molecular Weight Verification:

  • Confirm the detected band appears at the expected molecular weight of 23-25 kDa

  • Note that the calculated molecular weight is 23 kDa, while the observed molecular weight may be 23-25 kDa due to post-translational modifications

• Multiple Detection Methods:

  • Perform parallel validation using different techniques (WB, IHC, FC)

  • Use antibodies from different vendors or those targeting different epitopes

  • Compare results from both monoclonal (e.g., CST E7F4U or Bio-Rad OTI2E9 ) and polyclonal antibodies (e.g., Proteintech 10994-1-AP )

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • A specific antibody will show diminished or eliminated signal when blocked with its target peptide

  • For recombinant antibodies like CST's E7F4U, the immunizing sequence is described as "residues near the carboxy terminus of human ATP5O protein"

• Genetic Validation:

  • Use CRISPR/Cas9-mediated knockout of ATP5O

  • Employ RNA interference (RNAi) to reduce ATP5O expression

  • These methods should result in corresponding reduction or elimination of antibody signal

• Mass Spectrometry Correlation:

  • For ultimate validation, perform immunoprecipitation with the ATP5O antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm the presence of ATP5O peptides in the immunoprecipitate

This multi-faceted approach ensures high confidence in antibody specificity before proceeding with critical experiments, particularly important when investigating ATP5O's role in cancer biology where it has been identified as a potential biomarker for prostate and gastric cancers .

How can ATP5O antibodies be applied to investigate mitochondrial dysfunction in disease models?

ATP5O antibodies serve as powerful tools for investigating mitochondrial dysfunction in various disease models, particularly in cancer and metabolic disorders:

• Quantitative Expression Analysis:

  • Western Blot: Quantify ATP5O expression changes in disease versus control samples using recommended dilutions (1:500-1:3000)

  • Immunohistochemistry: Assess tissue-specific expression patterns in patient samples or animal models (1:50-1:500)

  • Flow Cytometry: Measure ATP5O levels at single-cell resolution (0.40 μg per 10^6 cells)

• Subcellular Localization Studies:

  • Immunofluorescence: Combine ATP5O antibodies with other mitochondrial markers to assess mitochondrial morphology and integrity

  • Subcellular Fractionation: Use ATP5O antibodies to confirm proper isolation of mitochondrial fractions

  • Super-resolution Microscopy: Investigate structural changes in mitochondrial ATP synthase organization

• Cancer Research Applications:

  • Monitor ATP5O expression in cancer-associated fibroblasts, where it has been shown to be elevated compared to normal fibroblasts

  • Investigate ATP5O as a biomarker in prostate and gastric cancers

  • Study the interaction between claudin-10 (CLDN10) and ATP5O, which has been shown to affect ATP5O expression and acetylation, leading to disrupted mitochondria and reduced metastasis in clear cell renal cell carcinoma

• Experimental Disease Models:

  • Metabolic Disorders: Assess ATP5O expression changes in models of diabetes, obesity, and mitochondrial diseases

  • Neurodegenerative Diseases: Investigate ATP5O alterations in Alzheimer's, Parkinson's, and ALS models

  • Ischemia/Reperfusion: Monitor ATP5O modifications during cellular stress conditions

• Therapeutic Response Monitoring:

  • Evaluate changes in ATP5O expression or post-translational modifications following treatment with mitochondria-targeting therapeutics

  • Use ATP5O as a marker for mitochondrial recovery in intervention studies

ATP5O antibodies enable researchers to connect changes in mitochondrial ATP synthase structure and function with disease progression, potentially identifying new therapeutic targets and biomarkers across multiple pathological conditions.

What are the optimal storage conditions for maintaining ATP5O antibody performance?

Proper storage of ATP5O antibodies is critical for maintaining their performance and extending their usable lifespan:

Following these storage guidelines will help ensure consistent results in applications including Western blot, immunohistochemistry, and flow cytometry when working with ATP5O antibodies from various manufacturers.

What controls should be included when investigating ATP5O with antibody-based techniques?

Proper experimental controls are essential for generating reliable and interpretable data when using ATP5O antibodies:

• Positive Controls:

  • Cell Lines: Include cells with known ATP5O expression such as A549, HEK-293, HeLa, HepG2, Caco-2, or HCT116 cells

  • Tissue Samples: Mouse heart, liver, placenta, and colon tissues have confirmed ATP5O expression

  • Human Tissues: For IHC, lung cancer tissue, colon cancer tissue, testis tissue, and heart tissue have been validated

• Negative Controls:

  • Primary Antibody Omission: Process samples without the primary ATP5O antibody

  • Isotype Controls: Use matched isotype control (e.g., rabbit IgG for polyclonal antibodies , mouse IgG1 for OTI2E9 clone )

  • Genetic Knockdown/Knockout: When available, include ATP5O-depleted samples

• Technique-Specific Controls:

  For Western Blot:

  • Loading controls such as GAPDH, β-actin, or preferably mitochondrial markers like VDAC

  • Molecular weight marker to confirm the expected 23-25 kDa band

  • Fractionation controls when isolating mitochondria (e.g., cytosolic marker)

  For Immunohistochemistry:

  • Serial sections with isotype control antibody

  • Known positive and negative tissue sections

  • Peptide competition (pre-absorption) controls

  For Flow Cytometry:

  • Unstained cells and isotype control

  • Permeabilization controls (ATP5O is intracellular)

  • Single-color controls for compensation when multiplexing

• Validation Controls:

  • Peptide Competition: Pre-incubate antibody with immunizing peptide

  • Multiple Antibodies: Use antibodies from different vendors targeting different epitopes

  • Alternative Detection Methods: Confirm findings using different techniques

• Experimental Condition Controls:

  • Treatment Groups: Include appropriate vehicle controls

  • Time Course: Sample at multiple time points to detect dynamic changes

  • Dose Response: Use multiple concentrations to establish specificity of effects

Implementing these controls will significantly enhance data quality and interpretation reliability when investigating ATP5O in research contexts ranging from basic mitochondrial biology to cancer biomarker studies.

How can researchers troubleshoot weak or absent ATP5O antibody signals?

When facing weak or absent ATP5O antibody signals, a systematic troubleshooting approach can help identify and resolve the issue:

• Sample Preparation Issues:

  • Protein Degradation: Ensure complete protease inhibitor cocktail usage during extraction

  • Insufficient Extraction: For mitochondrial proteins like ATP5O, standard RIPA buffer may be suboptimal; consider mitochondria-specific extraction buffers

  • Loading Amount: Increase protein loading (try 20-50 μg for whole cell lysates)

  • Sample Buffer: Verify SDS and reducing agent concentrations are adequate for complete denaturation

• Antibody-Related Factors:

  • Concentration: Try more concentrated primary antibody solutions (e.g., 1:500 instead of 1:3000 for WB)

  • Incubation Time: Extend primary antibody incubation (overnight at 4°C for WB, 48-72 hours for IHC)

  • Antibody Quality: Check expiration date and proper storage conditions (-20°C, avoid freeze-thaw cycles)

  • Alternative Antibody: Test another ATP5O antibody targeting a different epitope

• Detection System Optimization:

  • Enhanced Chemiluminescence: Use high-sensitivity ECL reagents for WB

  • Secondary Antibody: Verify species compatibility and increase concentration if needed

  • Exposure Time: Extend exposure time for WB detection

  • Signal Amplification: Consider tyramide signal amplification for IHC or ICC

• Application-Specific Troubleshooting:

  For Western Blot:

  • Transfer Efficiency: Check protein transfer with reversible staining

  • Membrane Type: PVDF membranes may provide better protein retention than nitrocellulose

  • Blocking Conditions: Excessive blocking can mask epitopes; try 3-5% BSA instead of milk for phospho-sensitive epitopes

  For Immunohistochemistry:

  • Antigen Retrieval: Test both recommended methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Fixation Issues: Overfixation can mask epitopes; consider reduced fixation time for future samples

  • Detection System: Switch to more sensitive detection system (e.g., polymer-based)

  For Flow Cytometry:

  • Permeabilization: Optimize permeabilization conditions for mitochondrial proteins

  • Antibody Amount: Increase to 0.80 μg per 10^6 cells (double the recommended amount)

  • Compensation: Ensure proper compensation if using multiple fluorophores

• Control Experiments:

  • Run positive control samples (e.g., HEK293, HeLa cells) alongside troubleshooting

  • Verify primary antibody function with dot blot of recombinant ATP5O

  • Test expression in tissues known to have high mitochondrial content (heart, liver)

By systematically addressing these factors, researchers can optimize ATP5O detection across multiple experimental platforms and obtain reliable, reproducible results.

What methods can be used to study ATP5O protein-protein interactions and complex formation?

Investigating ATP5O protein-protein interactions and its role in ATP synthase complex formation requires specialized techniques:

• Co-Immunoprecipitation (Co-IP):

  • Use ATP5O antibodies to pull down the protein and associated complex members

  • Western blot for known interaction partners (e.g., other ATP synthase subunits)

  • For optimal results, use mild lysis conditions to preserve native protein complexes

  • Consider crosslinking to stabilize transient interactions before immunoprecipitation

  • Example protocol: Use rabbit polyclonal antibodies (Proteintech 10994-1-AP) for immunoprecipitation followed by detection of interaction partners

• Proximity Ligation Assay (PLA):

  • Visualize and quantify protein-protein interactions in situ

  • Combine ATP5O antibodies with antibodies against potential interaction partners

  • Particularly useful for studying the interaction between ATP5O and claudin-10 (CLDN10), which has been shown to bind ATP5O in the outer mitochondrial membrane

  • Requires antibodies from different host species (e.g., rabbit anti-ATP5O and mouse anti-partner protein)

• Blue Native PAGE:

  • Preserve native protein complexes for size-based separation

  • Western blot with ATP5O antibodies to identify complex incorporation

  • Can reveal ATP5O distribution across different assembly intermediates of ATP synthase

  • Particularly valuable for studying how ATP5O connects the F1 catalytic domain and F0 membrane domain

• FRET/BRET Analysis:

  • For live-cell interaction studies with fluorescently tagged proteins

  • Generate fluorescent protein fusions with ATP5O and potential partners

  • Measure energy transfer as indication of protein proximity

  • Useful for dynamic interaction studies in response to metabolic changes

• Crosslinking Mass Spectrometry:

  • Chemical crosslinking of protein complexes followed by mass spectrometry

  • Identify direct binding partners of ATP5O

  • Map interaction interfaces at amino acid resolution

  • Can reveal novel interaction partners beyond known ATP synthase components

• Yeast Two-Hybrid or Mammalian Two-Hybrid:

  • Screen for novel interaction partners of ATP5O

  • Validate direct protein-protein interactions

  • Can identify domains involved in protein interactions

  • Consider using fragments of ATP5O to map interaction domains

• Microscopy-Based Approaches:

  • Super-resolution microscopy: Visualize ATP5O localization within mitochondrial structures

  • FRAP (Fluorescence Recovery After Photobleaching): Study ATP5O dynamics within the ATP synthase complex

  • Structured Illumination Microscopy (SIM): Observe ATP5O distribution in relation to cristae structure

These methodologies provide complementary approaches to understand ATP5O's role in ATP synthase assembly, its interactions with other mitochondrial proteins, and its contribution to cellular bioenergetics in normal and disease states.

How is ATP5O expression altered in cancer and what are the implications for using ATP5O antibodies in cancer research?

ATP5O expression undergoes significant alterations in cancer, making ATP5O antibodies valuable tools for cancer research:

• Expression Patterns in Cancer:

  • Elevated Expression: ATP5O is upregulated in cancer-associated fibroblasts (CAFs) compared to normal fibroblasts in oral squamous cell carcinoma

  • Biomarker Potential: Recent studies have identified elevated ATP5O as a biomarker for prostate and gastric cancers

  • Metastasis Connection: ATP5O expression and post-translational modifications influence metastatic potential in clear cell renal cell carcinoma (ccRCC)

• Application of ATP5O Antibodies in Cancer Research:

  • Tissue Screening: ATP5O antibodies have been validated for immunohistochemistry in various cancer tissues, including lung cancer and colon cancer

  • Prognostic Studies: Quantitative analysis of ATP5O expression using validated antibodies can help establish correlations with clinical outcomes

  • Mechanism Investigation: Antibodies enable study of ATP5O modifications and interactions that may drive cancer progression

• Regulation Mechanisms:

  • CLDN10 Interaction: Claudin-10 (CLDN10) binding to ATP5O in the outer mitochondrial membrane increases ATP5O expression and acetylation, disrupting mitochondria and reducing metastasis in ccRCC

  • Metabolic Reprogramming: Changes in ATP5O expression reflect metabolic adaptation in cancer cells and their microenvironment

  • Post-translational Modifications: Cancer-specific modifications of ATP5O may affect antibody recognition and function

• Experimental Approaches:

  • Expression Analysis: Compare ATP5O levels between tumor and adjacent normal tissues using Western blot (1:500-1:3000)

  • Tissue Microarrays: Assess ATP5O expression across cancer types and stages using immunohistochemistry (1:50-1:500)

  • Single-cell Analysis: Evaluate heterogeneity of ATP5O expression using flow cytometry (0.40 μg per 10^6 cells)

  • Functional Studies: Combine antibody-based detection with genetic manipulation of ATP5O to understand its mechanistic role

• Clinical Research Applications:

  • Patient Stratification: ATP5O expression patterns may help classify tumors into biologically relevant subtypes

  • Treatment Response: Monitor ATP5O as a potential indicator of response to mitochondria-targeting therapies

  • Drug Development: Use ATP5O antibodies to screen compounds that modulate its expression or function

The altered expression of ATP5O in various cancers highlights its potential as both a biomarker and therapeutic target. ATP5O antibodies provide essential tools for investigating these aspects, particularly given the protein's role in cellular bioenergetics and the metabolic reprogramming characteristic of cancer cells.

What experimental design considerations are important when using ATP5O antibodies in mitochondrial research?

When designing experiments using ATP5O antibodies for mitochondrial research, several critical factors must be considered:

• Experimental Model Selection:

  • Cell Lines: Choose models with appropriate mitochondrial content; HEK-293, HeLa, HepG2, and A549 cells have validated ATP5O expression

  • Tissue Samples: Heart, liver, and muscle tissues have high mitochondrial content and strong ATP5O expression

  • Disease Models: Select models relevant to mitochondrial dysfunction (e.g., neurodegenerative disease models, metabolic disorder models)

• Sample Preparation Optimization:

  • Mitochondrial Isolation: Consider subcellular fractionation to enrich for mitochondria

  • Preservation Methods: For immunohistochemistry, optimize fixation to preserve mitochondrial antigens

  • Lysis Conditions: Use mitochondria-appropriate lysis buffers that preserve ATP5O in its native conformation when needed

• Antibody Selection Strategy:

  • Application Matching: Choose antibodies validated for your specific application (WB, IHC, FC)

  • Epitope Consideration: Select antibodies targeting epitopes relevant to your research question

    • For studying N-terminal interactions with F1, choose antibodies targeting this region

    • For C-terminal interactions with F0, select antibodies targeting the C-terminus

  • Format Selection: Consider monoclonal antibodies for consistent results across experiments or polyclonal for signal amplification

• Controls and Validation:

  • Mitochondrial Markers: Include other mitochondrial proteins as controls (e.g., VDAC, COX IV)

  • Respiratory Chain Manipulation: Use inhibitors like oligomycin (which interacts with the ATP5O-associated region of ATP synthase)

  • Genetic Controls: Include ATP5O knockdown/knockout samples where possible

• Experimental Variables:

  • Metabolic State: Consider how different metabolic conditions affect ATP5O expression and complex formation

  • Stress Conditions: Examine ATP5O under relevant stressors (e.g., hypoxia, nutrient deprivation)

  • Dynamic Processes: Design time-course experiments to capture assembly/disassembly of ATP synthase complexes

• Advanced Applications:

  • Super-resolution Microscopy: For detailed localization studies, combine ATP5O antibodies with super-resolution techniques

  • Live-cell Imaging: Consider using ATP5O antibody fragments for live-cell applications

  • Multi-parameter Analysis: Design co-staining experiments with other mitochondrial markers for comprehensive analysis

• Data Interpretation Considerations:

  • Context-Specific Expression: ATP5O levels may vary significantly between tissues and cell types

  • Complex Assembly Status: ATP5O detection may reflect both free protein and complex-incorporated forms

  • Post-translational Modifications: Consider how phosphorylation at Y35, Y41, T43, and Y46 sites may affect results

By carefully addressing these experimental design considerations, researchers can maximize the utility of ATP5O antibodies in investigating mitochondrial structure, function, and dysfunction across various biological contexts.

How can flow cytometry with ATP5O antibodies be optimized for mitochondrial research?

Optimizing flow cytometry protocols for ATP5O detection requires specific considerations to account for its mitochondrial localization:

• Sample Preparation Protocol:

  • Cell Dissociation: Use gentle dissociation methods to preserve mitochondrial integrity

  • Fixation: 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilization Options:

    • Standard: 0.1-0.3% Triton X-100 (good for general mitochondrial proteins)

    • Enhanced: 0.05% saponin (gentler, may better preserve mitochondrial structure)

    • Alternative: 90% ice-cold methanol (excellent for detection of intracellular epitopes)

• Antibody Staining Optimization:

  • Concentration: Start with the recommended 0.40 μg per 10^6 cells in 100 μl suspension

  • Incubation Conditions: 30-60 minutes at room temperature or overnight at 4°C

  • Washing Buffer: PBS with 1-2% FBS or BSA to reduce background

  • Blocking: Include 5-10% normal serum from the secondary antibody species

• Controls for Accurate Analysis:

  • Unstained Cells: For autofluorescence assessment

  • Secondary-only Control: To establish background from secondary antibody

  • Isotype Control: Rabbit IgG at the same concentration as ATP5O antibody

  • Biological Controls: ATP5O-depleted cells (siRNA knockdown) or high-expression positive controls (HepG2 cells)

• Multi-parameter Analysis Design:

  • Mitochondrial Mass Markers: Combine with MitoTracker Green or VDAC antibodies

  • Mitochondrial Function Indicators: Include TMRE/JC-1 for membrane potential assessment

  • Cell Type Markers: Add lineage-specific surface markers for heterogeneous samples

• Data Acquisition Settings:

  • Compensation: Critical when combining ATP5O with other fluorescent markers

  • Gating Strategy:

    • Use FSC/SSC to select intact cells

    • Apply viability gating to exclude dead cells

    • Consider mitochondrial content gating for normalized analysis

• Analysis Approaches:

  • Mean Fluorescence Intensity (MFI): For quantitative comparison of ATP5O levels

  • Population Analysis: Identify subpopulations with distinct ATP5O expression

  • Correlation Analysis: Relate ATP5O levels to other mitochondrial parameters

• Advanced Applications:

  • Imaging Flow Cytometry: Combine quantitative data with localization information

  • Cell Sorting: Isolate populations based on ATP5O expression levels

  • Kinetic Analysis: Track changes in ATP5O levels following experimental treatments

• Troubleshooting Common Issues:

  • Low Signal: Increase antibody concentration, enhance permeabilization, or try alternative clones

  • High Background: Increase washing steps, optimize blocking, or reduce antibody concentration

  • Inconsistent Results: Standardize cell numbers, fixation time, and staining conditions