ATPAF1 Antibody

What is ATPAF1 and what role does it play in cellular function?

ATPAF1 (ATP synthase mitochondrial F1 complex assembly factor 1) is a nuclear-encoded protein essential for the assembly of the mitochondrial F1-F0 ATP synthase complex. It functions as a chaperone that specifically binds to the F1 beta subunit, preventing the formation of nonproductive homooligomers during enzyme assembly . The protein is critical for ATP synthesis and mitochondrial oxidative phosphorylation, playing a crucial role in cellular energy production .

ATPAF1 has a molecular weight of approximately 36 kDa (328 amino acids) as calculated, though the observed molecular weight in experimental conditions typically ranges from 31-35 kDa, likely due to post-translational modifications or processing .

What experimental applications are validated for ATPAF1 antibodies?

Most commercially available ATPAF1 antibodies have been validated for multiple applications, with varying degrees of optimization:

For optimal results, researchers should conduct preliminary titration experiments in their specific experimental systems, as sensitivity may vary depending on sample type and target expression levels .

What species reactivity can be expected with ATPAF1 antibodies?

The species reactivity profile varies among different ATPAF1 antibodies:

When working with non-human samples, researchers should verify cross-reactivity experimentally or select antibodies specifically validated for their species of interest .

What are the recommended protocols for ATPAF1 antibody use in immunohistochemistry?

For optimal immunohistochemistry results with ATPAF1 antibodies, the following protocol elements are critical:

• Antigen Retrieval: Tris-EDTA buffer (pH 9.0) is strongly recommended as the primary method. Citrate buffer (pH 6.0) can be used as an alternative but may yield lower sensitivity .

• Antibody Dilution: Start with a dilution range of 1:50-1:500, with most protocols suggesting 1:200 as an optimal starting point for paraffin-embedded tissue sections .

• Positive Controls: Human ovary cancer tissue and human stomach cancer tissue have been validated as reliable positive controls for ATPAF1 immunodetection .

• Detection System: An HRP-conjugated secondary antibody system typically provides good signal detection, as demonstrated in validation studies .

• Incubation Parameters: For primary antibody, incubation at 4°C overnight typically yields optimal results, followed by room temperature incubation with secondary antibody for 1 hour .

These parameters should be optimized for each specific experimental system to ensure reliable and reproducible results.

How should Western blot protocols be optimized for ATPAF1 detection?

For successful Western blot detection of ATPAF1, consider the following methodological details:

• Sample Preparation:

  • Total protein extraction from cells or tissues should be performed using standard lysis buffers containing protease inhibitors

  • Load 20-30 μg of total protein per lane for adequate detection

• Gel Electrophoresis:

  • 10% SDS-PAGE gels are appropriate for resolving ATPAF1 (31-36 kDa)

  • Include positive control samples (e.g., L02 cells, HEK-293 cells)

• Transfer and Blocking:

  • Standard PVDF or nitrocellulose membranes are suitable

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Primary antibody dilution: 1:500-1:2000 (optimal starting point: 1:1000)

  • Incubate at room temperature for 1.5 hours or at 4°C overnight

  • HRP-conjugated secondary antibody should be used at manufacturer-recommended dilutions

• Expected Band Size: Look for bands between 31-35 kDa depending on the specific antibody used

Validation data shows successful detection in various sample types including human cell lines (HEK-293, Raji, L02), mouse tissues (heart, kidney), and rat tissues (heart) .

How does ATPAF1 deficiency impact ATP synthase assembly and mitochondrial function?

Research using ATPAF1 knockout (KO) mouse models has revealed critical insights into the biological significance of this protein:

• ATP Synthase Assembly:

  • ATPAF1 deficiency leads to decreased F1 content and reduced ATP synthase dimers in cardiac tissue

  • Blue-Native electrophoresis (BN-PAGE) demonstrates impaired assembly of the complete ATP synthase complex

• Mitochondrial Structural Changes:

  • Electron microscopy reveals ultrastructural abnormalities in ATPAF1-deficient mitochondria

  • Key observations include condensed degenerated mitochondria and significant loss of cristae

• Functional Consequences:

  • Impaired respiratory capacity, particularly affecting complex V function

  • Elevated blood lactate levels, indicating a metabolic shift toward anaerobic glycolysis

  • Compromised autophagy and mitochondrial dynamics

• Physiological Impact:

  • ATPAF1-deficient mice have smaller body sizes

  • Develop decreased cardiac function in adulthood

  • Demonstrate that ATPAF1 is essential for maintaining cardiac structure and function

These findings highlight the critical role of ATPAF1 in energy metabolism and provide a model system for studying mitochondrial dysfunction in disease states.

What approaches can be used to study ATPAF1 interactions with ATP synthase components?

Several methodological approaches have been validated for investigating ATPAF1's interactions with the ATP synthase complex:

• Co-immunoprecipitation (Co-IP):

  • Successful Co-IP has been demonstrated using anti-ATP5B antibody with protein A/G magnetic beads

  • Reciprocal IP can be performed with tagged ATPAF1 constructs (e.g., ATPAF1-Flag)

  • Eluted complexes should be analyzed by SDS-PAGE and immunoblotting

• 2D Blue Native-SDS PAGE:

  • This technique separates intact ATP synthase complexes in the first dimension

  • Second dimension SDS-PAGE identifies individual components including ATPAF1

  • Western blotting with anti-ATPAF1 antibody can confirm its presence in specific complexes

• Protein Abundance Quantification:

  • emPAI (exponentially modified protein abundance index) calculations from mass spectrometry data can estimate relative abundance of ATPAF1 in different subcomplexes

  • This approach has revealed differential distribution patterns between monomeric and dimeric forms of ATP synthase

• Fluorescence Microscopy with Tagged Constructs:

  • Transfection of cells with ATPAF1-Flag constructs permits visualization of subcellular localization

  • Co-localization with ATP5B can be assessed using dual immunofluorescence

  • Mitochondrial markers (e.g., MitoTracker) can confirm mitochondrial targeting

These complementary approaches provide comprehensive insights into the physical and functional interactions of ATPAF1 within the ATP synthase assembly machinery.

What considerations are important when validating the specificity of ATPAF1 antibodies?

Rigorous validation of ATPAF1 antibodies is essential for reliable experimental outcomes. Consider these methodological approaches:

• Positive and Negative Control Samples:

  • Validated positive controls include L02 cells, HEK-293 cells, PC-3 cells for Western blot

  • For IHC, human ovary cancer tissue and human stomach cancer tissue serve as reliable positive controls

  • Consider using ATPAF1 knockout or knockdown samples as negative controls when available

• Cross-Reactivity Assessment:

  • Test multiple antibodies targeting different epitopes of ATPAF1

  • Compare reactivity patterns across different species (human, mouse, rat)

  • Evaluate potential cross-reactivity with related proteins through bioinformatic analysis

• Validation through Multiple Applications:

  • Confirm consistent results across different applications (WB, IHC, IF)

  • Verify expected molecular weight detection (31-36 kDa range)

  • Assess subcellular localization patterns consistent with mitochondrial distribution

• Literature Corroboration:

  • Compare results with published data using the same or different ATPAF1 antibodies

  • ATPAF1 antibody 18016-1-AP has been cited in at least 4 peer-reviewed publications, providing external validation

• Complementary Detection Methods:

  • Confirm findings using alternative approaches such as mass spectrometry

  • Use tagged ATPAF1 constructs (ATPAF1-Flag) for orthogonal validation

  • Consider mRNA expression data to correlate with protein expression patterns

Proper antibody validation enhances reproducibility and reliability of research findings involving ATPAF1.

What are common challenges in ATPAF1 antibody experiments and how can they be addressed?

Researchers may encounter several technical challenges when working with ATPAF1 antibodies:

• Variable Signal Intensity in Western Blots:

  • Problem: Weak or inconsistent band detection

  • Solution: Optimize protein loading (30 μg recommended), increase antibody concentration (1:500 instead of 1:1000), extend incubation time to overnight at 4°C, and use enhanced chemiluminescence detection systems

• Background in Immunohistochemistry:

  • Problem: High non-specific staining

  • Solution: Optimize blocking (increase to 5-10% serum or BSA), decrease primary antibody concentration (begin with 1:200 dilution), ensure proper antigen retrieval with Tris-EDTA buffer (pH 9.0), and include additional washing steps

• Epitope Masking:

  • Problem: Native ATPAF1 may not be recognized when incorporated into ATP synthase complexes

  • Solution: As observed in research, "anti-Mco10 antibody could not recognize the protein specifically in the ATP synthase monomer/dimer complexes - probably the peptide used for immunization is not exposed in the native Mco10"

  • Consider using denaturing conditions in Western blot and effective antigen retrieval in IHC

• Species Cross-Reactivity Issues:

  • Problem: Antibody may perform differently across species

  • Solution: Validate each antibody specifically for your species of interest; ATPAF1 antibody 15797-1-AP has demonstrated reactivity with human, mouse, and rat samples, making it suitable for comparative studies

How can researchers optimize subcellular localization studies of ATPAF1?

For accurate visualization of ATPAF1's subcellular distribution:

• Fixation and Permeabilization Optimization:

  • 4% formaldehyde fixation for 15 minutes at room temperature preserves structure

  • Permeabilization with 1% Triton X-100 for 15 minutes ensures antibody access to mitochondrial targets

• Co-localization Markers:

  • Include established mitochondrial markers (MitoTracker, ATP5B, or TOMM20)

  • ATP5B is particularly useful as it marks the same complex where ATPAF1 functions

  • Compare patterns with other mitochondrial proteins that may have differential distribution

• Advanced Microscopy Techniques:

  • Super-resolution microscopy can provide more detailed localization information

  • For dynamic studies, photoactivatable GFP (paGFP) fusion constructs allow tracking of newly synthesized protein

  • ATP5B-paGFP constructs have been successfully used to track ATP synthase movement and could be adapted for ATPAF1

• Mitochondrial Fraction Verification:

  • Complement imaging with biochemical fractionation to confirm mitochondrial enrichment

  • Western blot analysis of subcellular fractions can provide quantitative distribution data

  • The observed molecular weight of ATPAF1 (31-35 kDa) should be consistent across methods

These approaches collectively provide robust data on ATPAF1's localization and function within mitochondria.

How is ATPAF1 antibody research contributing to understanding mitochondrial dysfunction in disease?

ATPAF1 antibodies have enabled critical insights into mitochondrial pathology across multiple disease contexts:

• Cardiac Dysfunction:

  • ATPAF1 deficiency in mouse models leads to impaired cardiac function

  • Mitochondrial ultrastructural abnormalities include condensed degenerated mitochondria and loss of cristae

  • Western blot analysis with ATPAF1 antibodies demonstrates decreased F1 content in cardiac tissue

• Cancer Research:

  • ATPAF1 antibodies have been validated in ovarian and stomach cancer tissues

  • Immunohistochemistry protocols have been optimized for cancer tissue analysis

  • The relationship between ATP synthase assembly and cancer metabolism represents an emerging research area

• Metabolic Disorders:

  • ATPAF1 knockout mice exhibit elevated blood lactate levels, indicating metabolic dysfunction

  • Antibody-based detection methods allow monitoring of ATPAF1 expression in various metabolic conditions

  • Potential connections to diseases with mitochondrial etiology can be investigated using these tools

• Research Models:

  • CRISPR/Cas9-generated knockout mouse models have been developed and characterized

  • Antibody-based validation confirms the absence of ATPAF1 in these models

  • These models provide valuable platforms for studying mitochondrial dysfunction mechanisms

What methodological approaches can integrate ATPAF1 antibody data with functional mitochondrial assays?

Comprehensive mitochondrial research requires integration of multiple methodological approaches:

• Coordinated Protein Expression and Functional Analysis:

  • Western blot quantification of ATPAF1 levels can be correlated with ATP synthase activity assays

  • BN-PAGE analysis reveals ATP synthase assembly status which can be linked to respiratory capacity measurements

  • Compare results across different tissue types or disease models to establish functional relationships

• Microscopy-Function Integration:

  • Combine immunofluorescence microscopy with live-cell functional imaging

  • MitoTracker staining can assess mitochondrial membrane potential alongside ATPAF1 localization

  • Quantitative image analysis can correlate ATPAF1 distribution patterns with functional parameters

• Genetic Manipulation Studies:

  • Use ATPAF1 antibodies to validate knockdown or overexpression efficiency

  • Measure consequent changes in ATP synthase assembly and mitochondrial function

  • Rescue experiments with wild-type ATPAF1 in knockout models can confirm specificity of observed phenotypes

• Proteomic Approaches:

  • Immunoprecipitation with ATPAF1 antibodies followed by mass spectrometry identifies interaction partners

  • Changes in the ATP synthase interactome under different conditions can be analyzed

  • Data integration with functional assays provides mechanistic insights into mitochondrial dysfunction

By integrating these approaches, researchers can establish causative relationships between ATPAF1 expression, ATP synthase assembly, and mitochondrial function in health and disease.