atpE Antibody

What is atpE Antibody and what cellular processes does it target?

The atpE Antibody is a polyclonal antibody designed for research involving ATP synthase subunit c (atpE), a key component of F1F0 ATP synthase that plays a critical role in cellular energy metabolism. F-type ATPases consist of two structural domains, with the F1 domain containing the extramembraneous catalytic core. The atpE protein specifically corresponds to the subunit c component that helps produce ATP from ADP in the presence of a proton or sodium gradient. As a research tool, the antibody enables scientists to detect, quantify, and visualize this essential enzyme in various experimental contexts, providing insights into mitochondrial function and energy production pathways .

What are the main applications of atpE Antibody in research settings?

The atpE Antibody is validated for multiple research applications with distinct methodological approaches:

• ELISA (Enzyme-Linked Immunosorbent Assay): The primary validated application, useful for quantitative detection of atpE protein in solution-based samples

• Western blotting: For protein detection and semi-quantitative analysis of atpE in cell or tissue lysates

• Immunofluorescence: For visualization of atpE localization within cellular compartments, particularly mitochondria

• Immunohistochemistry: For detection of atpE in tissue sections to study distribution patterns

These techniques allow researchers to investigate ATP synthase expression levels, localization patterns, and potential alterations in various experimental conditions or disease models. The high specificity and sensitivity of the antibody make it particularly valuable for detecting subtle changes in energy metabolism pathways .

How should researchers prepare samples for optimal atpE Antibody binding?

For optimal atpE Antibody binding, researchers should follow these methodological steps:

• Sample preparation:

  • Cell cultures: Harvest cells at 70-80% confluence

  • Tissues: Flash-freeze and homogenize in appropriate buffer

  • Include protease inhibitors to prevent protein degradation

• Buffer composition:

  • Use a buffer compatible with the preservative (0.03% Proclin 300)

  • Maintain pH at 7.4 to match storage conditions

  • Include 50% glycerol for sample stability during longer procedures

• Blocking conditions:

  • Use 3-5% BSA or serum from a species different from the host (non-rabbit)

  • Block for 1-2 hours at room temperature to minimize background

• Temperature considerations:

  • Perform primary antibody incubation at 4°C overnight for best results

  • Allow reagents to reach room temperature before use

This methodological approach maximizes specific binding while reducing background, critical for accurate detection and quantification of atpE protein .

What species reactivity should researchers expect with commercial atpE Antibodies?

Researchers working with atpE Antibody should be aware of specific species reactivity limitations. According to technical specifications, the commercially available atpE Antibody (PACO35034) is specifically reactive to strain OhILAs ATP synthase subunit c protein. The antibody was generated using a recombinant strain OhILAs ATP synthase subunit c protein spanning amino acids 1-88 as the immunogen. This specific reactivity profile means:

• Primary reactivity: Confirmed for strain OhILAs samples

• Cross-reactivity: May potentially recognize homologous sequences in related bacterial strains, but this requires empirical validation

• Mammalian applications: Cross-reactivity with human, mouse, or other mammalian atpE homologs is not guaranteed and should be experimentally verified

Researchers should conduct preliminary validation studies when applying this antibody to species not explicitly listed in the reactivity profile, as sequence conservation in ATP synthase components varies across evolutionary distance .

How can researchers validate the specificity of atpE Antibody in their experimental systems?

Researchers should implement a multi-tiered validation approach to confirm atpE Antibody specificity:

• Molecular weight verification:

  • Run SDS-PAGE with positive and negative control samples

  • Confirm binding to proteins of expected molecular weight (~8-10 kDa for atpE)

  • Assess presence of non-specific bands

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (strain OhILAs ATP synthase subunit c, 1-88AA)

  • Compare results with and without peptide competition

  • Specific signals should be abolished or significantly reduced

• Knockout/knockdown controls:

  • Use CRISPR-Cas9 or RNAi to generate atpE-deficient cells

  • Compare antibody reactivity between wild-type and modified cells

  • Specific signals should be absent or reduced in modified samples

• Orthogonal detection methods:

  • Compare results with a second antibody targeting a different epitope

  • Correlate protein detection with mRNA expression data

  • Validate findings using mass spectrometry when possible

This comprehensive validation strategy ensures experimental observations genuinely reflect atpE biology rather than antibody artifacts .

What methodological approaches can enhance detection sensitivity when working with low-abundance atpE samples?

For enhanced detection of low-abundance atpE in biological samples, researchers can employ these methodological refinements:

• Signal amplification techniques:

  • Implement tyramide signal amplification (TSA) for immunohistochemistry applications

  • Use high-sensitivity chemiluminescent substrates for Western blotting

  • Consider polymer-based detection systems over conventional secondary antibodies

• Sample enrichment strategies:

  • Perform subcellular fractionation to isolate mitochondria-enriched fractions

  • Use immunoprecipitation to concentrate target protein before analysis

  • Implement ultracentrifugation techniques to separate membrane-associated proteins

• Optimized antibody parameters:

  • Extend primary antibody incubation time (up to 24-48 hours at 4°C)

  • Test concentration ranges above recommended dilutions

  • Evaluate signal-to-noise ratio at each concentration

• Instrument optimization:

  • Use highly sensitive CCD cameras for fluorescence detection

  • Optimize gain and exposure settings for quantitative imaging

  • Consider cooled-CCD systems to reduce background noise

These approaches maintain specificity while substantially improving detection limits for challenging samples .

How can atpE Antibody be utilized in the study of mitochondrial dysfunction and energy metabolic disorders?

atpE Antibody offers valuable research applications for investigating mitochondrial dysfunction through these methodological approaches:

• Differential expression analysis:

  • Compare atpE protein levels between normal and diseased tissues/cells

  • Correlate expression changes with functional mitochondrial assays

  • Implement quantitative Western blotting with normalization to mitochondrial mass markers

• Co-localization studies:

  • Perform dual immunofluorescence with markers of mitochondrial integrity

  • Assess potential redistribution of atpE during pathological states

  • Quantify co-localization coefficients using digital image analysis

• Post-translational modification assessment:

  • Combine atpE immunoprecipitation with mass spectrometry

  • Investigate modifications (phosphorylation, acetylation) affecting ATP synthase function

  • Develop modification-specific detection systems

• Pharmacological intervention studies:

  • Monitor atpE expression/localization in response to therapeutic compounds

  • Correlate molecular changes with functional recovery metrics

  • Develop high-throughput screening methods for compound effects on ATP synthase assembly

These approaches provide mechanistic insights into how ATP synthase alterations contribute to pathological conditions, potentially leading to novel therapeutic targets for mitochondrial disorders .

What are the critical controls needed when utilizing atpE Antibody in immunodetection experiments?

Implementing appropriate controls is essential for reliable atpE Antibody research. Researchers should incorporate the following controls based on experimental design:

• Antibody specificity controls:

  • Isotype control: Use non-specific rabbit IgG at identical concentration

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

  • Absorption control: Pre-incubate antibody with immunizing peptide

  • Positive control: Include sample known to express atpE (strain OhILAs)

• Sample preparation controls:

  • Loading control: Validate equal protein loading using housekeeping proteins

  • Subcellular fraction purity: Confirm mitochondrial enrichment with markers like COX IV

  • Degradation assessment: Evaluate sample integrity with broad-spectrum protein stains

• Methodological controls:

  • Technical replicates: Perform multiple measurements to assess reproducibility

  • Dilution series: Confirm signal linearity across sample concentrations

  • Inter-assay calibrators: Include standard samples across experiments for normalization

• Data analysis controls:

  • Blinded quantification: Analyze images without knowledge of sample identity

  • Signal saturation test: Ensure signal falls within linear detection range

  • Background subtraction validation: Apply consistent background definition across all samples

This comprehensive control framework enables confident interpretation of experimental results and identification of potential methodological artifacts .

How should researchers optimize protein extraction protocols for maximal recovery of atpE protein?

Optimizing extraction of atpE protein requires specialized approaches due to its membrane-associated nature and relatively small size. Researchers should implement this methodological workflow:

• Lysis buffer composition optimization:

  • Include non-ionic detergents (0.5-1% Triton X-100 or NP-40)

  • Add mild ionic detergents (0.1-0.5% sodium deoxycholate)

  • Incorporate chaotropic agents (1-2M urea) for membrane disruption

  • Maintain physiological pH (7.2-7.6) to preserve protein structure

• Physical disruption techniques:

  • For tissues: Utilize mechanical homogenization followed by sonication

  • For cells: Apply multiple freeze-thaw cycles with sonication

  • Consider nitrogen cavitation for gentle membrane disruption

• Solubilization enhancement:

  • Heat samples to 60-70°C (not boiling) in SDS-containing buffer

  • Allow extended incubation times (30-60 minutes) for complete solubilization

  • Use increasing detergent gradients to identify optimal concentration

• Protein preservation strategies:

  • Include comprehensive protease inhibitor cocktail

  • Perform all extraction steps at 4°C

  • Process samples immediately after collection

  • Avoid repeated freeze-thaw cycles

This optimized approach maximizes recovery of atpE protein while maintaining its antigenic properties for subsequent immunodetection .

What considerations should guide antibody dilution optimization for atpE detection?

Systematic optimization of antibody dilution is critical for balancing sensitivity and specificity in atpE detection. Researchers should follow this methodological approach:

• Initial dilution range determination:

  • Start with manufacturer's recommended dilution (based on application)

  • Prepare a logarithmic dilution series spanning at least 3-fold above and below

  • For ELISA applications, include 2-fold dilution steps for finer optimization

• Signal-to-noise evaluation:

  • Calculate signal-to-noise ratio for each dilution

  • Plot dilution versus signal-to-noise to identify optimal range

  • Determine minimum dilution that produces acceptable background

• Application-specific considerations:

  • Western blotting: Optimize based on protein load (10-50 μg total protein)

  • Immunofluorescence: Consider tissue type and fixation method

  • ELISA: Adjust based on antigen concentration and detection system

• Validation of optimized dilution:

  • Confirm reproducibility with independent samples

  • Verify linearity of signal across relevant concentration range

  • Assess inter-lot consistency if using multiple antibody lots

For the atpE Antibody (PACO35034), researchers should note that while general dilution recommendations exist, optimal dilutions must be determined empirically for each experimental system, as factors like sample preparation, detection method, and instrument sensitivity significantly impact performance .

How can researchers address non-specific binding when using atpE Antibody?

Non-specific binding presents a significant challenge when working with atpE Antibody. Researchers can implement these methodological solutions:

• Blocking optimization:

  • Test alternative blocking agents (5% BSA, 5% non-fat milk, commercial blockers)

  • Extend blocking time to 2-3 hours at room temperature

  • Include 0.1-0.3% Tween-20 in blocking solution to reduce hydrophobic interactions

  • Consider fish gelatin-based blockers for highly sensitive applications

• Washing protocol enhancement:

  • Increase washing stringency with higher salt concentration (up to 500 mM NaCl)

  • Extend wash duration and increase wash steps (5-6 washes of 10 minutes each)

  • Add low concentrations of SDS (0.01-0.05%) to wash buffers for membrane applications

  • Implement temperature-controlled washing (4°C for reduced dissociation, 37°C for increased stringency)

• Antibody incubation modification:

  • Pre-adsorb antibody with tissue/cell lysate from species of interest

  • Dilute antibody in buffer containing 1-5% of species-matched normal serum

  • Add non-ionic detergents (0.1-0.3% Triton X-100) to antibody diluent

  • Consider overnight incubation at 4°C rather than shorter room temperature incubation

• Sample preparation refinement:

  • Implement additional purification steps for complex samples

  • Consider protein A/G pre-clearing to remove endogenous immunoglobulins

  • Use freshly prepared samples to minimize denaturation and aggregation

These interventions should be systematically tested and documented to establish an optimized protocol for specific experimental conditions .

What analytical approaches can help distinguish true atpE signal from potential artifacts?

Distinguishing genuine atpE signals from artifacts requires rigorous analytical approaches:

• Multi-technique validation:

  • Confirm findings using at least two independent detection methods

  • Compare results between different antibody-based techniques (Western blot vs. IF vs. ELISA)

  • Correlate protein detection with mRNA expression data where possible

• Quantitative analysis frameworks:

  • Implement digital image analysis with consistent thresholding parameters

  • Use ratiometric analysis comparing target to internal controls

  • Apply statistical approaches to distinguish signal from background variation

  • Consider machine learning algorithms for pattern recognition in complex images

• Biological validation strategies:

  • Correlate atpE signals with functional measurements of ATP synthesis

  • Assess co-localization with other mitochondrial markers

  • Verify expected response patterns to known modulators of ATP synthase

• Technical artifact elimination:

  • Implement blind duplicate analysis by independent researchers

  • Document consistent findings across multiple experimental replicates

  • Develop criteria for exclusion of technically compromised samples

  • Consider natural signal distribution patterns across tissue/cell types

How should researchers interpret unexpected molecular weight bands when using atpE Antibody in Western blotting?

Unexpected molecular weight bands in atpE Western blotting require systematic interpretation:

• Biological source assessment:

  • Post-translational modifications: Phosphorylation, glycosylation, or ubiquitination may increase apparent molecular weight

  • Protein-protein interactions: Incomplete denaturation may preserve complexes

  • Alternative splicing: Verify against known splice variants in sequence databases

  • Proteolytic fragments: May indicate sample degradation or physiological processing

• Technical artifact evaluation:

  • Non-specific binding: Test with blocking peptide competition

  • Cross-reactivity: Conduct bioinformatic analysis for sequence similarity with detected bands

  • Secondary antibody binding: Perform secondary-only control

  • Sample preparation issues: Evaluate different extraction and denaturation methods

• Verification experiments:

  • Immunoprecipitation followed by mass spectrometry

  • Comparison with antibodies targeting different epitopes

  • siRNA/CRISPR knockdown to confirm specificity

  • Subcellular fractionation to localize unexpected bands

• Quantitative band analysis:

  • Document relative intensity of expected versus unexpected bands

  • Assess reproducibility across different samples and experiments

  • Investigate biological conditions that alter band patterns

The expected molecular weight for atpE (ATP synthase subunit c) is approximately 8-10 kDa, though oligomeric forms may appear at higher molecular weights. Bands at significantly different weights warrant careful investigation using the approaches outlined above .

How can atpE Antibody be utilized in multiparameter imaging studies?

atpE Antibody can be incorporated into sophisticated multiparameter imaging through these methodological approaches:

• Multiplexed immunofluorescence strategies:

  • Spectral unmixing: Utilize fluorophores with distinct emission spectra

  • Sequential detection: Employ multiple rounds of staining with intermediate stripping

  • Multispectral imaging: Capture spectral signatures across wavelength ranges

  • Combine with tyramide signal amplification for improved sensitivity

• Correlative microscopy techniques:

  • Combine fluorescence with electron microscopy for ultrastructural context

  • Implement super-resolution methods (STORM, PALM, STED) for nanoscale localization

  • Register light and electron microscopy images for multi-scale analysis

  • Use fiducial markers for precise alignment of imaging modalities

• Live-cell imaging applications:

  • Generate cell lines expressing fluorescently tagged atpE for dynamic studies

  • Correlate fixed-cell antibody staining with live-cell functional imaging

  • Implement reversible membrane permeabilization for antibody delivery to live cells

  • Use proximity ligation assays to detect protein-protein interactions in situ

• Spatial analysis frameworks:

  • Quantify co-localization coefficients with mitochondrial markers

  • Analyze morphological parameters of atpE-positive structures

  • Implement neighborhood analysis for spatial relationships

  • Develop 3D reconstruction of atpE distribution patterns

These advanced imaging approaches provide unprecedented insights into atpE distribution, dynamics, and functional interactions within the complex cellular environment .

What considerations should guide researchers in selecting between different aqueous two-phase extraction (ATPE) systems for antibody purification?

When selecting appropriate ATPE systems for antibody purification, researchers should consider these methodological factors:

• Partition coefficient optimization:

  • PEG/phosphate systems offer excellent recovery for antibodies

  • Adjust molecular weight of PEG (4000-8000 Da) based on antibody characteristics

  • Modify phase-forming salt concentration to optimize partition

  • Consider pH effects on antibody charge and partitioning behavior

• Process integration factors:

  • Continuous ATPE systems incorporating extraction, back-extraction, and washing improve purity

  • Microfluidic platforms allow rapid screening of multiple extraction conditions

  • Pump mixer-settler batteries enable scale-up from laboratory to production

  • Evaluate compatibility with upstream and downstream processing steps

• Impurity removal efficiency:

  • Higher molecular weight impurities typically separate during extraction

  • Lower molecular weight impurities require subsequent back-extraction and washing

  • Evaluate host cell protein removal across different ATPE systems

  • Consider DNA and endotoxin removal requirements

• Scale-up and economic considerations:

  • Assess reagent costs and recyclability of phase components

  • Evaluate process throughput and continuous processing potential

  • Consider equipment requirements and capital investment

  • Factor in validation and reproducibility across scales

This systematic approach to ATPE system selection ensures optimal performance for specific antibody purification requirements .