ATPA Antibody

What is an ATPA antibody and what cellular components does it target?

ATPA antibodies are immunoglobulins that specifically recognize and bind to ATP synthase alpha subunit or other ATP-related proteins. The most common targets include ATP citrate lyase (ACLY), ATP synthase components, and ATPase phospholipid transporting proteins such as ATP8A2. These antibodies are crucial for studying energy metabolism, mitochondrial function, and lipid synthesis pathways in cells. ATP citrate lyase, for instance, is a 1188-amino acid protein belonging to the Cation transport ATPase (P-type) family, with membrane-associated cellular localization . When selecting an ATPA antibody, researchers should consider the specific target protein and its subcellular localization to ensure appropriate experimental design and interpretation.

What are the primary applications for ATPA antibodies in research?

ATPA antibodies are versatile tools employed across multiple experimental techniques. Based on the search results, the most common applications include:

These applications enable researchers to detect, quantify, and localize ATP-related proteins in various sample types, providing insights into energy metabolism pathways and mitochondrial function . When designing experiments, consider that some antibodies may perform better in specific applications than others, as indicated by supplier validation data.

How should I select the appropriate ATPA antibody for my research?

Selecting the right ATPA antibody requires consideration of several key factors:

• Target specificity: Determine exactly which ATP-related protein you need to detect. Search results show multiple targets including ATP citrate lyase, ATP synthase components, and ATP transporters .

• Species reactivity: Verify that the antibody recognizes your experimental species. Many antibodies show cross-reactivity with human, mouse, and rat proteins, but species-specific variations exist .

• Application compatibility: Ensure the antibody is validated for your intended application. For example, if performing immunofluorescence, select antibodies explicitly validated for IF use .

• Clonality: Consider whether monoclonal or polyclonal antibodies are more suitable for your research. Monoclonal antibodies like [EP704Y] offer high specificity, while polyclonal antibodies may provide stronger signals through multiple epitope recognition .

• Citation record: Review publications that have used the antibody successfully. The search results indicate some antibodies have substantial publication records, with some having over 100 figures across numerous publications .

A methodical approach to antibody selection increases experimental reliability and reproducibility. When possible, test multiple antibodies against your specific samples to determine optimal performance.

What controls should I include when working with ATPA antibodies?

Proper controls are essential for interpreting results obtained with ATPA antibodies:

• Positive control: Include samples known to express your target ATP protein. For ATP citrate lyase antibodies, liver tissue or HepG2 cells typically express high levels and serve as excellent positive controls .

• Negative control: Use samples where the target is absent or knocked down. For ATP synthase studies, mitochondria-depleted cells can serve as negative controls .

• Isotype control: Include an irrelevant antibody of the same isotype, concentration, and host species to detect non-specific binding.

• Blocking peptide control: When available, pre-incubate the antibody with its specific antigenic peptide to confirm signal specificity.

• Loading controls: For western blotting, include housekeeping proteins (β-actin, GAPDH) to normalize target protein expression.

• Secondary antibody-only control: Omit primary antibody to detect non-specific secondary antibody binding.

Including these controls helps distinguish genuine signals from artifacts and strengthens the reliability of your research findings. Documentation of controls is particularly important when investigating post-translational modifications of ATP proteins, such as phosphorylation of ATP citrate lyase at Ser455 .

How can I optimize detection of phosphorylated ATP citrate lyase with phospho-specific antibodies?

Detecting phosphorylated ATP citrate lyase (pACLY) requires specific optimization strategies:

• Sample preparation: Rapidly harvest cells or tissues and immediately lyse in buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails) to preserve phosphorylation status.

• Antibody selection: Use phospho-specific antibodies targeting key regulatory sites, such as Ser455, which has been extensively validated across 95 figures from 69 publications according to search results . This site is particularly informative as it reflects the activation state of ATP citrate lyase.

• Stimulation conditions: To detect dynamic changes in phosphorylation, compare basal conditions with stimulated conditions. EGF stimulation has been reported to increase pACLY levels with clear band differentiation when using appropriate antibodies .

• Dephosphorylated controls: Treat duplicate samples with lambda phosphatase before immunoblotting to confirm phospho-specificity of the antibody.

• Signal enhancement: For weak signals, consider using enhanced chemiluminescence substrates or signal amplification systems compatible with your detection method.

• Quantification method: Use densitometry to quantify the ratio of phosphorylated to total ATP citrate lyase, normalizing to loading controls for accurate comparison between treatment conditions.

For reliable results, always validate phospho-specific antibodies in your experimental system using positive controls such as cells treated with growth factors known to induce ATP citrate lyase phosphorylation .

What are the key considerations for subcellular localization studies of ATP synthase using immunofluorescence?

Visualizing ATP synthase localization requires careful experimental design:

• Fixation method: Different ATP synthase subunits may require specific fixation protocols. Paraformaldehyde (4%) is commonly used, but some epitopes may be better preserved with methanol fixation, particularly for mitochondrial components.

• Permeabilization: Since ATP synthase is primarily mitochondrial, ensure adequate permeabilization (0.1-0.5% Triton X-100 or 0.1% saponin) to allow antibody access to mitochondrial membranes without destroying organelle integrity.

• Co-localization markers: Include established mitochondrial markers (MitoTracker, TOMM20 antibody) to confirm proper localization of ATP synthase signals.

• Z-stack imaging: Collect confocal microscopy z-stacks to properly visualize the three-dimensional distribution of mitochondria and associated ATP synthase complexes.

• Super-resolution approaches: Consider techniques like STED or STORM microscopy for distinguishing ATP synthase localization on inner mitochondrial membrane cristae versus other mitochondrial compartments.

• Live-cell imaging: For dynamic studies, consider antibody fragments or fluorescently tagged ATP synthase constructs for live-cell visualization of ATP synthase distribution during cellular processes.

Researchers should be aware that non-canonical localizations of ATP synthase components have been reported outside mitochondria in specific cell types, requiring careful validation of unexpected localization patterns .

How can I resolve contradictory results when using different ATPA antibodies in my experiments?

Resolving conflicting results requires systematic troubleshooting:

• Epitope mapping: Determine the epitope regions recognized by each antibody. Antibodies targeting different domains of the same protein may give different results if:

  • Post-translational modifications mask specific epitopes

  • Protein conformation varies between experimental conditions

  • Protein interactions shield certain epitopes

• Validation approach:

  • Perform knockdown/knockout validation for each antibody

  • Test antibodies on recombinant protein standards

  • Compare results with orthogonal detection methods (mass spectrometry)

• Cross-reactivity assessment: Evaluate potential cross-reactivity with related proteins. For example, some ATP synthase antibodies may cross-react with different subunits or isoforms .

• Protocol optimization for each antibody:

  • Adjust antibody concentration

  • Modify blocking conditions

  • Alter incubation times and temperatures

  • Test different detection systems

• Sample preparation variables:

  • Ensure consistent sample processing

  • Compare different lysis buffers

  • Standardize protein quantification methods

• Independent verification: Consult published literature for consensus on expected results. The search results show that some antibodies have extensive publication records with consistent findings across multiple studies , providing benchmarks for comparison.

When reporting contradictory results, transparently document all methodological differences that might account for the discrepancies, including antibody catalog numbers, dilutions, and detailed protocols.

What strategies can optimize co-immunoprecipitation of ATP synthase complexes while maintaining native interactions?

Co-immunoprecipitation (Co-IP) of ATP synthase complexes requires preserving delicate protein-protein interactions:

• Lysis buffer optimization:

  • Use mild, non-denaturing detergents (0.5-1% digitonin, 0.5-1% CHAPS, or 0.1-0.5% NP-40)

  • Include physiological salt concentration (150mM NaCl)

  • Maintain neutral pH (7.2-7.4)

  • Add protease inhibitors freshly before use

• Antibody selection criteria:

  • Choose antibodies validated for immunoprecipitation applications

  • Select clones that recognize epitopes not involved in protein-protein interactions

  • Consider using antibodies against tags if working with tagged versions of ATP synthase components

• Bead selection:

  • Magnetic beads often provide gentler handling than agarose

  • Pre-clear lysates to reduce non-specific binding

  • Optimize antibody-to-bead ratio for maximum efficiency

• Washing conditions:

  • Use multiple gentle washes with decreasing detergent concentrations

  • Maintain consistent temperature during washing steps

  • Consider including stabilizing agents like glycerol or ATP in wash buffers

• Elution strategies:

  • Competitive elution with peptides for gentlest approach

  • Acidic glycine buffer (pH 2.5-3.0) with immediate neutralization

  • SDS elution for maximum recovery but potential complex disruption

• Verification approaches:

  • Blue native PAGE to confirm intact complexes

  • Mass spectrometry to identify all interacting partners

  • Reciprocal Co-IP with antibodies against suspected interaction partners

Researchers should note that ATP synthase exists in supercomplexes with other mitochondrial proteins, requiring particularly careful handling to maintain these higher-order interactions during immunoprecipitation procedures .

How can I quantitatively assess ATP synthase activity in conjunction with antibody-based detection methods?

Integrating functional and expression analyses provides comprehensive insights:

For robust analysis, combine enzymatic activity assays with quantitative immunoblotting of ATP synthase subunits to correlate protein expression levels with functional output. This approach can reveal whether alterations in ATP production stem from changes in protein abundance, post-translational modifications, or intrinsic catalytic efficiency .

What are the critical considerations when using ATPA antibodies for quantitative tissue profiling across different pathological conditions?

Tissue profiling with ATPA antibodies requires standardized approaches:

• Sample collection standardization:

  • Match cases and controls for age, sex, and relevant clinical parameters

  • Standardize time from tissue collection to fixation/freezing

  • Document tissue handling protocols thoroughly for reproducibility

• Antibody validation for tissue specificity:

  • Test antibody performance on tissue microarrays containing relevant tissue types

  • Include positive and negative control tissues in each experiment

  • Verify staining patterns with multiple independent antibodies when possible

• Staining optimization for quantification:

  • Develop consistent antigen retrieval protocols

  • Standardize antibody concentration through titration experiments

  • Ensure linear range of detection for quantitative analysis

• Image acquisition parameters:

  • Maintain identical exposure settings across all samples

  • Collect sufficient fields of view for statistical significance

  • Include internal reference standards in each imaging session

• Quantification approach:

  • Define objective scoring criteria (H-score, digital image analysis)

  • Perform blinded scoring by multiple observers

  • Validate quantification with orthogonal approaches (e.g., comparing IHC with proteomics)

• Data interpretation considerations:

  • Account for tissue heterogeneity in analysis

  • Consider cell type-specific expression patterns

  • Correlate expression with relevant clinical outcomes

When analyzing ATP citrate lyase or ATP synthase expression across disease states, researchers should be particularly attentive to metabolic variations between tissues, as these proteins play central roles in energy metabolism and may show context-dependent regulation .

How can I address non-specific background when using ATPA antibodies in immunohistochemistry?

Background reduction requires systematic optimization:

• Primary antibody optimization:

  • Titrate antibody concentration to determine optimal dilution

  • Test different diluents (PBS-T, TBS-T with 1-5% BSA or normal serum)

  • Extend primary antibody incubation (overnight at 4°C) with more dilute antibody

• Blocking optimization:

  • Increase blocking agent concentration (3-10% normal serum from secondary antibody species)

  • Extend blocking time (1-2 hours at room temperature)

  • Add protein blockers (1-5% BSA, casein, or commercial protein blocks)

  • Include avidin/biotin blocking for biotin-based detection systems

• Tissue preparation improvements:

  • Optimize fixation time for formalin-fixed tissues

  • Refine antigen retrieval conditions (pH, temperature, duration)

  • Include peroxidase quenching step (0.3-3% H₂O₂ for 10-30 minutes)

• Washing modifications:

  • Increase number and duration of washes

  • Add detergent (0.05-0.1% Tween-20) to wash buffers

  • Use high-salt wash buffers (up to 500mM NaCl) for high-background antibodies

• Detection system considerations:

  • Test polymer-based vs. traditional secondary antibody systems

  • Consider tyramide signal amplification for weak signals with minimal background

  • Optimize chromogen development time with close monitoring

For ATP synthase or ATP citrate lyase antibodies, researchers should be particularly attentive to mitochondria-rich tissues where endogenous biotin or peroxidase activity may contribute to background signals .

What approaches can resolve weak or inconsistent signals when detecting ATP citrate lyase phosphorylation?

Enhancing phosphorylation-specific signals requires targeted strategies:

• Sample preservation optimization:

  • Harvest samples rapidly to prevent phosphatase activity

  • Use buffer with phosphatase inhibitor cocktail plus 50mM NaF and 1mM Na₃VO₄

  • Process all samples at 4°C to minimize dephosphorylation

• Signal enhancement techniques:

  • Use high-sensitivity ECL substrates for western blotting

  • Apply tyramide signal amplification for IHC/IF detection

  • Consider biotin-streptavidin amplification systems

• Phospho-enrichment approaches:

  • Perform phosphoprotein enrichment before detection

  • Use titanium dioxide or immobilized metal affinity chromatography

  • Apply phosphopeptide immunoprecipitation for mass spectrometry validation

• Experimental design adjustments:

  • Include positive control samples (EGF-stimulated cells show clear pACLY bands)

  • Create time-course experiments to capture peak phosphorylation

  • Compare multiple lysis methods to optimize epitope accessibility

• Antibody optimization:

  • Test multiple phospho-specific antibodies targeting the same site

  • Use antibody cocktails to enhance signal strength

  • Extend primary antibody incubation time (overnight at 4°C)

When working specifically with Phospho-ATP-Citrate Lyase (Ser455) antibodies, researchers report cleaner band detection with reduced background when using optimized blocking conditions and extended washing protocols .

How should I validate ATPA antibodies for cross-reactivity and specificity in my experimental system?

Comprehensive validation ensures reliable research outcomes:

• Genetic validation approaches:

  • Test antibody on knockout/knockdown samples

  • Use CRISPR-modified cell lines with epitope mutations

  • Compare signals across species with known sequence divergence

• Biochemical validation methods:

  • Perform peptide competition assays with immunizing peptide

  • Test reactivity against recombinant protein fragments

  • Conduct epitope mapping with peptide arrays

• Orthogonal detection techniques:

  • Compare antibody results with mass spectrometry data

  • Validate subcellular localization with fractionation studies

  • Correlate protein detection with mRNA expression data

• Cross-reactivity assessment:

  • Test on samples expressing related family members

  • Perform western blots to confirm single band of expected size

  • Conduct immunoprecipitation followed by mass spectrometry

• Functional correlation:

  • Link antibody detection with known biological functions

  • Verify response to stimuli known to affect the target protein

  • Correlate with activity assays where applicable

For ATP citrate lyase antibodies specifically, validation against phospho-ATP-Citrate Lyase (Ser455) can be performed by treating cells with EGF, which induces phosphorylation at this site, followed by detection with phospho-specific antibodies .

How can ATPA antibodies be effectively utilized in multiplexed imaging systems?

Multiplexed detection enables comprehensive spatial analysis:

• Fluorescence multiplexing approaches:

  • Select primary antibodies from different host species

  • Use directly conjugated primary antibodies with non-overlapping fluorophores

  • Employ sequential antibody detection with stripping or quenching between rounds

• Mass cytometry and imaging mass cytometry:

  • Use metal-conjugated ATPA antibodies for highly multiplexed detection

  • Design panel with other metabolic and signaling markers

  • Analyze spatial relationships between ATP synthase and other proteins

• Cyclic immunofluorescence (CycIF):

  • Incorporate ATPA antibodies in multi-round staining protocols

  • Optimize fluorophore inactivation between cycles

  • Develop computational pipelines for image registration and analysis

• Proximity ligation assays:

  • Study protein-protein interactions between ATP synthase components

  • Investigate spatial relationships between ATP synthetic machinery and substrates

  • Quantify interaction frequencies in different cellular contexts

• Multiplex chromogenic IHC:

  • Utilize spectrally distinct chromogens for ATP synthase subunits

  • Develop sequential chromogenic detection protocols

  • Apply computational pathology for quantitative analysis

When designing multiplexed panels including ATP synthase or ATP citrate lyase antibodies, researchers should consider the subcellular localization of targets to ensure appropriate visualization and quantification strategies .

What considerations are important when using ATPA antibodies for super-resolution microscopy studies?

Super-resolution imaging requires specialized antibody considerations:

• Fluorophore selection criteria:

  • Choose bright, photostable fluorophores compatible with super-resolution technique

  • For STORM/PALM, select fluorophores with appropriate blinking characteristics

  • For STED, use fluorophores with high depletion efficiency

• Sample preparation optimization:

  • Use thin samples to minimize spherical aberration

  • Optimize fixation to preserve nanoscale structures

  • Consider expansion microscopy for improved resolution of mitochondrial structures

• Antibody format considerations:

  • Use smaller detection probes (Fab fragments, nanobodies) for improved resolution

  • Consider direct primary antibody labeling to eliminate displacement error

  • Test site-specific labeling strategies for consistent fluorophore positioning

• Validation approaches:

  • Correlate super-resolution with electron microscopy

  • Perform dual-label experiments with established markers

  • Use complementary super-resolution techniques for confirmation

• Quantitative analysis strategies:

  • Develop cluster analysis workflows for ATP synthase distribution

  • Measure nearest-neighbor distances between complexes

  • Quantify colocalization at nanometer precision

For ATP synthase studies, super-resolution microscopy has revealed important insights into the nanoscale organization of ATP synthase dimers along mitochondrial cristae, information not accessible with conventional microscopy .