ATP5D Antibody

What is ATP5D and why is it important in cellular research?

ATP5D is a subunit of 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. This gradient is generated by electron transport complexes of the respiratory chain. The importance of ATP5D lies in its role as a key component of the F1 domain containing the extramembraneous catalytic core of ATP synthase. During catalysis, ATP turnover in the catalytic domain of F1 is coupled via a rotary mechanism of the central stalk subunits to proton translocation . Recent research has revealed that ATP5D is particularly significant in cancer cell metabolism, as it is involved in the regulation of glycolysis through RNA m1A methylation mechanisms .

What applications are ATP5D antibodies validated for?

ATP5D antibodies have been validated for multiple research applications with varying levels of optimization:

The most commonly validated applications include Western Blot (WB), Immunohistochemistry on paraffin-embedded tissues (IHC-P), and Immunocytochemistry/Immunofluorescence (ICC/IF) . When designing experiments, researchers should select the antibody that has been specifically validated for their application of interest and target species.

What is the recommended protocol for using ATP5D antibodies in immunohistochemistry?

For paraffin-embedded tissue sections, a validated protocol involves the following methodology:

• Deparaffinize and rehydrate tissue sections through a graded alcohol series

• Perform antigen retrieval using TrilogyTM (EDTA-based, pH 8.0) buffer for 15 minutes

• Block endogenous peroxidase and non-specific binding sites

• Apply ATP5D antibody (e.g., GTX101503) at a dilution of 1:250

• Incubate at 4°C overnight

• Apply appropriate secondary antibody and develop using a detection system

• Counterstain, dehydrate, and mount

This protocol has been demonstrated to effectively detect ATP5D protein at mitochondria in human breast cancer tissue samples . Optimization may be necessary for different tissue types, fixation methods, or when using alternative antibody clones.

How can I optimize Western blot protocols for ATP5D detection?

ATP5D has a calculated molecular weight of approximately 17.5 kDa , which requires specific optimization for reliable detection:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for optimal protein extraction

  • Sonicate samples briefly to ensure complete lysis of mitochondrial membranes

  • Heat samples at 70°C rather than 95°C to prevent aggregation of membrane proteins

• Gel electrophoresis:

  • Use 12-15% polyacrylamide gels for better resolution of smaller proteins

  • Load 20-30 μg of total protein per lane

• Transfer and detection:

  • Transfer at lower voltage (30V) overnight at 4°C for efficient transfer of small proteins

  • Use PVDF membrane with 0.2 μm pore size rather than 0.45 μm

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

  • Incubate with primary ATP5D antibody at 1:500-1:2000 dilution

  • Use high-sensitivity detection reagents

This optimized protocol accounts for the small size and membrane-association properties of ATP5D, ensuring more consistent and specific detection.

What controls should be included when using ATP5D antibodies?

Appropriate controls are critical for interpreting ATP5D antibody results:

• Positive controls:

  • HeLa cell lysate (human cervical cancer cell line)

  • SiHa cell lysate (human cervical squamous cell line)

  • Tissues with known high ATP5D expression (e.g., breast cancer tissue)

• Negative controls:

  • Primary antibody omission control

  • Non-specific IgG control at equivalent concentration

  • ATP5D knockdown or knockout samples (ALKBH3-/- HeLa cells show decreased ATP5D levels)

  • Non-expressing tissues or cell lines (specific to experimental context)

• Loading controls:

  • Mitochondrial markers (e.g., VDAC1, COX IV) for fraction purity

  • Standard housekeeping proteins (e.g., GAPDH, β-actin) for total protein normalization

Inclusion of these controls enables proper validation of signal specificity and appropriate quantitative analysis.

How does ATP5D differ from other ATP synthase subunits in research applications?

While ATP synthase is composed of multiple subunits, ATP5D has several distinct characteristics that make it uniquely valuable for specific research applications:

• Size distinction: At approximately 17.5 kDa , ATP5D is smaller than many other ATP synthase subunits, allowing for different experimental approaches when studying protein complexes.

• Functional uniqueness: ATP5D is part of the F1 catalytic domain and plays a specific role in the rotary mechanism coupling ATP synthesis to proton translocation .

• Post-translational modifications: ATP5D is subject to specific regulation by RNA m1A methylation that affects its translation, making it a valuable target for studying post-transcriptional regulation mechanisms .

• Cancer metabolism connections: Research has specifically linked ATP5D to glycolysis in cancer cells, positioning it as a potential marker for metabolic reprogramming in tumors .

When designing experiments targeting ATP synthase, researchers should consider whether their scientific question specifically relates to ATP5D's functions or would be better addressed by targeting other subunits or the complex as a whole.

What is the relationship between ATP5D and cancer cell metabolism?

Recent research has revealed a complex relationship between ATP5D and cancer cell metabolism, particularly through epigenetic regulatory mechanisms:

• Glycolytic regulation: ATP5D has been found to positively regulate glycolysis in cancer cells. Overexpression of ATP5D can increase glucose consumption, lactate production rate, and ATP levels in cancer cells .

• Methylation control: ATP5D expression is regulated by m1A RNA methylation. The m1A modified A71 at exon 1 of ATP5D negatively regulates its translation elongation by increasing binding with the YTHDF1/eRF1 complex, which facilitates the release of mRNA from the ribosome complex .

• Demethylase influence: The m1A demethylase ALKBH3 regulates ATP5D expression in a demethylation-dependent manner. In ALKBH3 knockout cells, ATP5D protein levels decrease significantly, correlating with reduced glycolytic activity .

• Transcriptional regulation: m1A also regulates mRNA stability of E2F1, which directly binds to the ATP5D promoter to initiate its transcription, creating a multi-level regulatory network .

These findings suggest that ATP5D could be a potential target for cancer therapeutic strategies aimed at metabolic reprogramming.

What are the most common challenges when using ATP5D antibodies, and how can they be addressed?

Researchers frequently encounter several specific challenges when working with ATP5D antibodies:

• Background signal in mitochondria-rich tissues:

  • Solution: Use more stringent blocking (2% BSA + 5% normal serum)

  • Optimize antibody concentration through careful titration

  • Consider antigen retrieval optimization for IHC applications

• Cross-reactivity with other ATP synthase subunits:

  • Solution: Select antibodies raised against unique epitopes (e.g., C-terminal region)

  • Validate specificity using knockout controls

  • Perform peptide competition assays with the immunizing peptide

• Variable detection in different sample types:

  • Solution: Adjust lysis protocols for different tissues (brain vs. muscle vs. cancer)

  • Optimize protein extraction for membrane-associated proteins

  • Consider using mitochondrial enrichment protocols for low-abundance samples

• Inconsistent results across applications:

  • Solution: Select application-specific validated antibodies

  • Optimize fixation and permeabilization for ICC/IF applications

  • Adjust exposure times for weaker signals in Western blots

Addressing these challenges through methodological optimization enhances experimental reproducibility and data reliability.

How can I use ATP5D antibodies to study its role in RNA methylation-dependent translation?

To investigate the role of ATP5D in RNA methylation-dependent translation, researchers can implement the following experimental approach:

• Methylation status analysis:

  • Perform m1A-seq to identify methylation sites within the ATP5D mRNA

  • Use m1A antibody for RNA immunoprecipitation (RIP) followed by qPCR to quantify methylation levels

  • Compare m1A levels in normal vs. ALKBH3 knockout cells

• Translation efficiency assessment:

  • Conduct polysome profiling to isolate translation-active ribosomes

  • Perform qPCR to quantify ATP5D mRNA in different ribosomal fractions

  • Compare the distribution of ATP5D mRNA in monosome vs. polysome fractions between control and ALKBH3-deficient cells

• RNA-protein interaction analysis:

  • Use CLIP-qPCR to assess binding between ATP5D mRNA and translation factors (YTHDF1, eIF4E, eEF1, eEF2, eRF1, eRF3)

  • Compare binding efficiency between wild-type and ALKBH3 knockout conditions

  • Perform RNA-protein pull-down assays with synthesized methylated and unmethylated ATP5D RNA fragments

• Functional validation:

  • Create reporter constructs with ATP5D coding sequences fused to luciferase

  • Measure translation efficiency using dual-luciferase assays

  • Introduce targeted demethylation using dm1ACRISPR systems to validate methylation-specific effects

This comprehensive approach enables detailed mechanistic understanding of how m1A methylation affects ATP5D expression and subsequent metabolic outcomes.

How can ATP5D antibodies be used to investigate mitochondrial dysfunction in disease models?

ATP5D antibodies can serve as powerful tools for investigating mitochondrial dysfunction across various disease models:

• Neurodegenerative diseases:

  • Use immunofluorescence with ATP5D antibodies to assess mitochondrial morphology and distribution in neuronal cultures

  • Combine with markers of oxidative stress to correlate ATP synthase integrity with damage

  • Compare ATP5D levels and localization between healthy and diseased brain tissues

• Metabolic disorders:

  • Quantify ATP5D expression changes in insulin-resistant tissues

  • Correlate ATP5D levels with ATP production capacity in muscle biopsies

  • Assess ATP5D incorporation into ATP synthase complexes using blue native PAGE followed by Western blotting

• Cancer metabolism reprogramming:

  • Compare ATP5D expression across cancer subtypes with different metabolic profiles

  • Correlate ATP5D levels with glycolytic markers and patient outcomes

  • Use ATP5D antibodies in combination with phospho-specific antibodies to assess metabolic signaling pathway activation

• Aging research:

  • Track age-dependent changes in ATP5D expression across tissues

  • Correlate with markers of mitochondrial quality control

  • Investigate post-translational modifications using specific antibodies combined with ATP5D detection

These applications leverage ATP5D antibodies as markers for both the integrity and functionality of the ATP synthase complex in pathological conditions.

What methodologies combine ATP5D antibodies with other techniques for comprehensive mitochondrial analysis?

Innovative multi-modal approaches integrating ATP5D antibody detection with other techniques provide deeper insights into mitochondrial biology:

• Super-resolution microscopy with ATP5D antibodies:

  • STORM or PALM imaging to visualize ATP5D distribution within single mitochondria

  • Dual-color imaging with inner membrane markers to assess ATP synthase organization

  • Live-cell compatible antibody fragments for dynamic ATP synthase tracking

• Proximity labeling with ATP5D antibodies:

  • BioID or APEX2 fusion proteins to identify proximal interacting partners

  • Combined with mass spectrometry to map the ATP5D interaction network

  • Temporal analysis to detect dynamic interaction changes during cellular stress

• Integrated omics approaches:

  • Correlate ATP5D protein levels (detected via antibodies) with transcriptomic data

  • Link post-translational modifications (detected by modification-specific antibodies) with metabolomic profiles

  • Create integrated models of ATP synthase regulation across multiple molecular levels

• Functional respiratory analysis with ATP5D quantification:

  • Seahorse XF analysis of oxygen consumption rate (OCR) combined with ATP5D immunodetection

  • Correlation of extracellular acidification rate (ECAR) with ATP5D expression levels

  • Real-time monitoring of mitochondrial membrane potential with parallel ATP5D quantification

These integrated approaches overcome the limitations of antibody-only detection methods and provide contextual information about ATP5D's role in mitochondrial function.