PET100 Antibody

What is PET100 and why is it significant in research?

PET100 is a mitochondrial protein that functions as a cytochrome c oxidase chaperone. With a molecular weight of approximately 9.1 kDa and 73 amino acid residues in humans, it plays a vital role in the assembly and maturation of mitochondrial complex IV (cytochrome c oxidase). The significance of PET100 in research stems from its association with mitochondrial complex IV deficiency, a condition that affects cellular energy production. Understanding PET100's function provides insights into mitochondrial diseases and potential therapeutic approaches. The protein is conserved across species, with orthologs reported in mouse, rat, bovine, frog, zebrafish, and chimpanzee, making it suitable for comparative studies of mitochondrial function .

What types of PET100 antibodies are available for research applications?

Researchers have access to several types of PET100 antibodies, including:

• Polyclonal antibodies produced in rabbit, such as HPA067288, which are suitable for immunofluorescence applications

• Non-conjugated antibodies for ELISA, immunofluorescence (IF), and immunohistochemistry (IHC) applications with human reactivity

• Conjugated antibodies, including:

  • Biotin-conjugated antibodies for ELISA

  • FITC-conjugated antibodies for ELISA and fluorescence-based applications

These antibodies can be used to detect human PET100, and some are available for detecting PET100 in other species including bacteria and Saccharomyces .

What is the recommended subcellular localization analysis method for PET100?

For subcellular localization analysis of PET100, immunofluorescence microscopy is the recommended method. The protein is primarily localized in the mitochondria, and proper analysis requires:

• Using anti-PET100 antibodies at a concentration of 0.25-2 μg/mL for optimal staining

• Co-staining with established mitochondrial markers (e.g., MitoTracker or anti-COX IV antibodies)

• High-resolution confocal microscopy to confirm the mitochondrial localization pattern

• Comparison with the known punctate mitochondrial staining pattern documented in the Human Protein Atlas

This approach allows for accurate determination of PET100's subcellular distribution and can help identify any mislocalization in disease models or experimental conditions .

How should PET100 antibodies be validated for experimental use?

Thorough validation of PET100 antibodies is critical before experimental use. A comprehensive validation protocol includes:

• Western blot analysis: Confirm antibody specificity by detecting a single band at approximately 9.1 kDa in mitochondrial fractions.

• Preadsorption assays: Use recombinant PET100 protein (such as APrEST94289) as a blocking agent to confirm antibody specificity. The antigen sequence ELWPPEKLQEIEEFKERLRKRREEKLLRDAQQ can be used for preadsorption .

• Positive and negative controls:

  • Positive control: Tissues or cells known to express PET100 (e.g., mitochondria-rich tissues)

  • Negative control: PET100 knockout cells or tissues, or samples treated with siRNA against PET100

• Cross-reactivity testing: If working with non-human samples, test the antibody against the species of interest, as PET100 orthologs exist in mouse, rat, bovine, frog, zebrafish, and chimpanzee .

• Application-specific controls: For each application (ELISA, IF, IHC), include appropriate isotype controls and secondary antibody-only controls.

This validation approach ensures reliable and reproducible results in downstream applications .

What is the optimal protocol for immunofluorescence staining using PET100 antibodies?

For optimal immunofluorescence staining with PET100 antibodies, follow this detailed protocol:

• Sample preparation:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum (matching secondary antibody host) in PBS for 1 hour

  • Incubate with anti-PET100 antibody at 0.25-2 μg/mL in blocking buffer overnight at 4°C

  • Wash 3x with PBS, 5 minutes each

  • Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

  • Counterstain with DAPI for nuclear visualization

• Mitochondrial co-staining (recommended):

  • Include mitochondrial markers such as MitoTracker or antibodies against other mitochondrial proteins

  • This confirms the expected mitochondrial localization of PET100

• Controls:

  • Include a sample incubated with the PrEST antigen PET100 (APrEST94289) to demonstrate specificity

  • Include secondary antibody-only controls

• Imaging parameters:

  • Use confocal microscopy for optimal resolution of mitochondrial structures

  • Capture z-stacks to fully visualize three-dimensional mitochondrial networks

This protocol optimizes detection while minimizing background and ensuring specificity of the staining pattern .

How should PET100 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of PET100 antibodies are essential for maintaining their activity and extending their usable life:

• Storage conditions:

  • Store antibodies at -20°C as recommended by manufacturers

  • Avoid repeated freeze-thaw cycles; aliquot antibodies upon receipt

  • For working solutions, store at 4°C for up to two weeks

• Handling guidelines:

  • Thaw antibodies completely before use and mix gently by inversion

  • Avoid vortexing, which can damage antibody structure

  • Centrifuge briefly before opening to collect solution at the bottom

  • Use sterile techniques when handling to prevent contamination

• Buffer considerations:

  • Anti-PET100 antibodies are typically supplied in buffered aqueous glycerol solutions

  • Do not dilute stock antibody unless preparing working solutions

  • For dilutions, use buffers recommended by the manufacturer

• Shipping and temporary storage:

  • If antibodies arrive on wet ice, transfer immediately to -20°C storage

  • During experiments, keep antibodies on ice when not in use

Following these guidelines ensures maximum antibody performance and reproducibility across experiments .

How can PET100 antibodies be utilized to study mitochondrial complex IV deficiency?

PET100 antibodies can be powerful tools for investigating mitochondrial complex IV deficiency through several advanced approaches:

• Patient-derived cell analysis:

  • Compare PET100 expression and localization in patient-derived fibroblasts versus healthy controls

  • Assess correlation between PET100 levels and complex IV activity using combined immunostaining and enzymatic assays

• Co-immunoprecipitation studies:

  • Use PET100 antibodies to pull down protein complexes

  • Identify PET100 interaction partners in normal versus disease states

  • Map the PET100 interactome during complex IV assembly

• Time-course expression analysis:

  • Monitor PET100 expression during mitochondrial biogenesis

  • Correlate PET100 levels with complex IV assembly stages

  • Investigate compensatory mechanisms in complex IV deficiency models

• Therapeutic screening platforms:

  • Develop high-content screening assays using PET100 antibodies to identify compounds that restore proper PET100 function or localization

  • Validate hits with functional assays of complex IV activity

• Structure-function studies:

  • Combine immunolabeling with site-directed mutagenesis of PET100

  • Identify critical domains for proper localization and function

This multi-faceted approach provides comprehensive insights into the role of PET100 in mitochondrial complex IV deficiency pathogenesis and potential therapeutic avenues .

What are the considerations for designing multiplex experiments using PET100 antibodies?

Designing effective multiplex experiments with PET100 antibodies requires careful planning to ensure compatibility and meaningful results:

• Antibody compatibility assessment:

  • Ensure primary antibodies are raised in different host species to prevent cross-reactivity

  • If using multiple rabbit antibodies (including anti-PET100), consider sequential staining protocols with intermediate blocking steps

  • Verify that secondary antibodies do not cross-react with non-target primary antibodies

• Fluorophore selection strategy:

  • Choose fluorophores with minimal spectral overlap (e.g., FITC, TRITC, Cy5)

  • Consider conjugated primary antibodies where available, such as FITC or biotin-conjugated PET100 antibodies

  • Account for relative expression levels of target proteins when selecting fluorophore brightness

• Co-localization analysis design:

  • Pair PET100 with other mitochondrial proteins (e.g., COX subunits, VDAC, TOM20)

  • Include markers for mitochondrial dynamics or quality control (e.g., DRP1, MFN2, PINK1)

  • Use super-resolution microscopy for detailed co-localization studies

• Controls for multiplex experiments:

  • Single-stain controls for each antibody to establish baseline signals

  • Isotype controls for each primary antibody used

  • Compensation controls if using flow cytometry applications

• Quantification approaches:

  • Establish protocols for measuring co-localization coefficients

  • Develop pipelines for high-throughput image analysis

  • Use appropriate statistical methods for analyzing multi-parameter data

This structured approach enables complex experimental designs while maintaining specificity and quantitative rigor .

How can PET100 antibodies be used to investigate the relationship between mitochondrial dysfunction and disease pathogenesis?

PET100 antibodies offer valuable tools for exploring the connections between mitochondrial dysfunction and disease mechanisms:

• Disease model characterization:

  • Profile PET100 expression in neurodegenerative disease models (e.g., Parkinson's, Alzheimer's)

  • Investigate PET100 alterations in metabolic disorders and cancer models

  • Correlate PET100 expression with mitochondrial morphology and function in disease states

• Tissue-specific mitochondrial assessment:

  • Compare PET100 expression across tissues with varying metabolic demands

  • Investigate tissue-specific consequences of PET100 mutations

  • Correlate PET100 levels with tissue-specific manifestations of mitochondrial disease

• Stress response studies:

  • Monitor PET100 levels during oxidative stress, hypoxia, or nutrient deprivation

  • Assess the relationship between PET100 and mitochondrial quality control pathways

  • Investigate whether PET100 serves as an early marker for mitochondrial dysfunction

• Therapeutic response monitoring:

  • Use PET100 immunostaining to track mitochondrial recovery after intervention

  • Assess whether PET100 levels correlate with functional improvement

  • Develop PET100-based biomarkers for treatment efficacy

• Combined approaches:

  • Integrate PET100 immunodetection with metabolomic analysis

  • Correlate PET100 alterations with changes in cellular bioenergetics

  • Combine with genetic approaches to identify modifiers of PET100 function

These research strategies facilitate comprehensive investigation of mitochondrial dysfunction in disease pathogenesis, potentially revealing new therapeutic targets .

What are common issues when using PET100 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with PET100 antibodies. Here are common issues and their solutions:

• Weak or absent signal:

  • Cause: Insufficient antibody concentration, degraded antibody, or low target expression

  • Solution: Increase antibody concentration (try 2 μg/mL for immunofluorescence), verify storage conditions, and use positive control samples known to express PET100

• High background staining:

  • Cause: Insufficient blocking, antibody concentration too high, or non-specific binding

  • Solution: Extend blocking time, optimize antibody dilution, include 0.1% Tween-20 in wash buffers, and use the recommended diluent

• Non-specific mitochondrial staining:

  • Cause: Cross-reactivity with other mitochondrial proteins

  • Solution: Perform preadsorption controls with the recombinant PET100 antigen (APrEST94289), and validate with knockout or knockdown controls

• Inconsistent results between experiments:

  • Cause: Variations in fixation, permeabilization, or antibody handling

  • Solution: Standardize protocols, prepare aliquots of antibody to avoid freeze-thaw cycles, and include internal controls in each experiment

• Discrepancy between techniques:

  • Cause: Different epitope accessibility in various applications

  • Solution: Validate the antibody separately for each application (WB, IF, IHC), and consider using different antibodies optimized for specific techniques

• Poor co-localization with mitochondrial markers:

  • Cause: Suboptimal fixation or permeabilization affecting epitope accessibility

  • Solution: Test different fixation methods (paraformaldehyde vs. methanol) and adjust permeabilization conditions

These troubleshooting approaches ensure reliable and reproducible results when working with PET100 antibodies .

How can researchers ensure batch-to-batch consistency when using PET100 antibodies?

Ensuring batch-to-batch consistency is crucial for longitudinal studies and reproducible research with PET100 antibodies:

• Reference sample benchmarking:

  • Maintain a reference sample (cell lysate or tissue) with known PET100 expression

  • Test each new antibody batch against this reference to establish comparable working dilutions

  • Document signal intensity and pattern for standardization

• Standardized validation protocol:

  • Develop a consistent validation workflow for each new antibody lot

  • Include Western blot for specificity, immunofluorescence for localization pattern, and ELISA for sensitivity

  • Maintain a validation report template for systematic comparison

• Internal controls implementation:

  • Include positive and negative controls in each experiment

  • Use recombinant PET100 protein standards for quantitative applications

  • Consider including an invariant protein control (e.g., GAPDH) for normalization

• Lot-specific optimization:

  • Titrate each new antibody lot to determine optimal working concentration

  • Document lot-specific differences in sensitivity and background

  • Adjust protocols accordingly while maintaining consistent experimental conditions

• Long-term reference material storage:

  • Create and store a large batch of reference samples (cells/lysates)

  • Aliquot and store at -80°C to minimize freeze-thaw cycles

  • Use these references throughout the project lifetime

By implementing these strategies, researchers can minimize variability introduced by antibody lot changes and ensure consistent, reliable results in their PET100 research .

How should experiments be designed to study PET100's role in mitochondrial complex IV assembly?

Designing experiments to elucidate PET100's role in mitochondrial complex IV assembly requires a multifaceted approach:

• Genetic manipulation strategies:

  • Generate PET100 knockdown/knockout models using siRNA or CRISPR-Cas9

  • Create rescue experiments with wild-type and mutant PET100 constructs

  • Develop inducible expression systems for temporal control of PET100 expression

• Interaction analysis framework:

  • Perform co-immunoprecipitation using anti-PET100 antibodies to identify binding partners

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

  • Employ BioID or APEX2 proximity labeling with PET100 fusion proteins

• Assembly kinetics assessment:

  • Design pulse-chase experiments with newly synthesized mitochondrial proteins

  • Use synchronized cells to track complex IV assembly during the cell cycle

  • Implement time-course analysis of PET100 association with assembly intermediates

• Structural analysis approach:

  • Create domain deletion and point mutation variants of PET100

  • Assess the impact on complex IV assembly using anti-PET100 antibodies

  • Correlate structural alterations with functional outcomes

• Quantitative metrics design:

  • Establish assays for complex IV activity (cytochrome c oxidation)

  • Develop quantitative immunofluorescence protocols using calibrated standards

  • Implement blue native PAGE to visualize assembly intermediates

This comprehensive experimental design allows researchers to systematically investigate PET100's specific role in the complex IV assembly pathway and identify critical functional domains .

What are the key considerations when designing Western blot experiments with PET100 antibodies?

Western blotting for PET100 requires specific considerations due to its small size (9.1 kDa) and mitochondrial localization:

• Sample preparation optimization:

  • Use mitochondrial enrichment protocols to concentrate the target protein

  • Include protease inhibitors to prevent degradation of the small protein

  • Consider native versus denaturing conditions based on experimental goals

• Gel electrophoresis parameters:

  • Use high percentage (15-20%) polyacrylamide gels for optimal resolution of the 9.1 kDa protein

  • Consider Tricine-SDS-PAGE systems designed for small proteins

  • Use appropriate molecular weight markers that include the low MW range

• Transfer conditions adjustment:

  • Optimize transfer time and voltage for small proteins (typically lower voltage for longer time)

  • Consider semi-dry transfer systems which can be more efficient for small proteins

  • Use PVDF membranes with 0.2 μm pore size rather than 0.45 μm for better retention

• Antibody selection and validation:

  • Confirm that the selected anti-PET100 antibody works in Western blot applications

  • Use the PrEST antigen PET100 (APrEST94289) as a positive control

  • Include preadsorption controls to confirm specificity

• Detection system considerations:

  • Choose high-sensitivity detection methods for this low-abundance protein

  • Consider enhanced chemiluminescence or fluorescence-based detection

  • Optimize exposure times to capture the small protein band without overexposure

• Controls and normalization:

  • Include positive controls (samples with known PET100 expression)

  • Use appropriate loading controls (preferably mitochondrial proteins of similar size)

  • Consider using total protein normalization methods for more accurate quantification

These specialized considerations help overcome the challenges associated with Western blotting of small mitochondrial proteins like PET100 .

How can researchers implement multiplexed analysis workflows that incorporate PET100 antibodies?

Implementing multiplexed analysis workflows with PET100 antibodies enables comprehensive mitochondrial studies:

• Multiparameter flow cytometry design:

  • Combine PET100 antibodies with markers for mitochondrial mass, membrane potential, and ROS

  • Use fixation and permeabilization protocols optimized for mitochondrial proteins

  • Implement compensation controls for each fluorophore combination

• High-content imaging workflow:

  • Design automated imaging protocols that capture PET100 alongside other mitochondrial proteins

  • Incorporate live-cell probes for functional parameters (e.g., MitoTracker, TMRE)

  • Develop image analysis pipelines to extract multi-parameter data from single cells

• Mass cytometry (CyTOF) implementation:

  • Label anti-PET100 antibodies with rare earth metals

  • Combine with antibodies against other mitochondrial proteins and cellular markers

  • Create analytical workflows to visualize complex relationships between parameters

• Spatial proteomics integration:

  • Combine PET100 immunostaining with RNA in situ hybridization

  • Implement multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX)

  • Analyze spatial relationships between PET100 and other mitochondrial components

• Multi-omics approach:

  • Correlate PET100 protein levels (immunoassays) with transcriptomics data

  • Integrate with metabolomics to assess functional consequences of PET100 alterations

  • Develop computational pipelines to analyze integrated datasets

Table 1: Recommended Multiplexed Panel Design for Mitochondrial Research Using PET100 Antibodies

This table provides a foundation for designing multiplexed experiments that position PET100 within the broader context of mitochondrial structure and function .

How should researchers interpret contradictory results between different detection methods for PET100?

When faced with contradictory results between different detection methods for PET100, a systematic approach to interpretation and troubleshooting is necessary:

• Method-specific epitope accessibility analysis:

  • Different detection methods expose different epitopes

  • Western blot denatures proteins, potentially exposing epitopes hidden in native conformation

  • Immunofluorescence preserves spatial context but may restrict access to some epitopes

  • Consider using multiple antibodies targeting different PET100 epitopes for validation

• Technical parameters evaluation:

  • Assess fixation impacts: Paraformaldehyde versus methanol fixation can yield different results

  • Evaluate detergent effects: Different permeabilization reagents may affect epitope recognition

  • Consider buffer conditions: pH and salt concentration can influence antibody-epitope interactions

• Expression level threshold considerations:

  • Western blot may detect bulk protein levels that immunofluorescence cannot visualize

  • Immunofluorescence may detect localized concentrations missed in whole-cell lysates

  • Quantify signal-to-noise ratios for each method to establish detection limits

• Integrated data interpretation framework:

  • Develop a decision tree for weighing contradictory results

  • Prioritize results from methods with more extensive validation

  • Consider orthogonal approaches (e.g., mass spectrometry) to resolve contradictions

• Biological versus technical variability assessment:

  • Determine if contradictions reflect true biological differences

  • Evaluate technical reproducibility within each method

  • Design experiments to distinguish between biological and technical factors

By systematically evaluating these factors, researchers can resolve contradictions and develop a more nuanced understanding of PET100 expression and function across experimental contexts .

What analytical approaches are recommended for quantifying PET100 expression in different experimental contexts?

Quantifying PET100 expression accurately requires tailored analytical approaches for different experimental contexts:

• Western blot quantification:

  • Use densitometry with appropriate software (ImageJ, Image Lab)

  • Implement standard curves with recombinant PET100 protein

  • Normalize to mitochondrial loading controls (e.g., VDAC, TOM20)

  • Apply rolling ball background subtraction for small proteins

  • Report relative expression as fold-change from control samples

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity within mitochondrial regions

  • Use automated segmentation of mitochondria based on specific markers

  • Apply background subtraction using non-mitochondrial regions

  • Analyze cell-to-cell variability with single-cell analysis

  • Consider 3D quantification with z-stack confocal images

• ELISA/immunoassay analysis:

  • Develop standard curves using recombinant PET100 protein

  • Apply four-parameter logistic regression for curve fitting

  • Calculate absolute concentration from standard curves

  • Assess coefficients of variation for technical and biological replicates

  • Validate using spike-and-recovery experiments

• Flow cytometry analysis:

  • Gate on intact, single cells before PET100 analysis

  • Use median fluorescence intensity rather than mean for robustness

  • Apply compensation matrices for multiplexed experiments

  • Consider ratio metrics (PET100/mitochondrial mass) for normalization

  • Analyze population heterogeneity with visualization tools

• Transcriptomics integration:

  • Correlate protein levels with mRNA expression

  • Calculate protein-to-mRNA ratios to assess post-transcriptional regulation

  • Use regression analysis to identify factors affecting expression correlation

These quantification approaches provide rigorous and reproducible measures of PET100 expression that can be compared across experimental conditions and between studies .

What emerging technologies might enhance PET100 antibody-based research?

Several cutting-edge technologies are poised to revolutionize PET100 antibody-based research:

• Single-molecule localization microscopy:

  • Super-resolution techniques (PALM, STORM) can resolve individual PET100 molecules

  • Reveals nanoscale organization within mitochondrial subcompartments

  • Enables precise quantification of molecular clustering and distribution

• Expansion microscopy:

  • Physical expansion of samples improves resolution with standard microscopes

  • Allows visualization of PET100 distribution within mitochondrial cristae

  • Compatible with multiplexed protein detection for contextual analysis

• Spatially-resolved proteomics:

  • Combines imaging with mass spectrometry for in situ protein identification

  • Provides comprehensive protein landscape surrounding PET100

  • Reveals spatial organization of mitochondrial complexes

• Antibody engineering advances:

  • Single-domain antibodies (nanobodies) for improved penetration and resolution

  • Site-specific conjugation for precise fluorophore positioning

  • Bifunctional antibodies that can simultaneously detect PET100 and interacting partners

• Live-cell antibody applications:

  • Cell-permeable antibody fragments for real-time imaging

  • Antibody-based biosensors to detect PET100 conformational changes

  • Optogenetic antibody systems for light-controlled manipulation

• Artificial intelligence integration:

  • Deep learning for automated image analysis and pattern recognition

  • Machine learning algorithms to identify subtle phenotypic changes

  • Predictive modeling of PET100 interactions and functions

These emerging technologies will enable researchers to address previously intractable questions about PET100 dynamics, interactions, and functions in normal and disease states .

How might PET100 antibody research contribute to understanding broader mitochondrial disease mechanisms?

PET100 antibody research has significant potential to advance our understanding of mitochondrial disease mechanisms:

• Assembly pathway mapping:

  • Using PET100 antibodies to trace the stepwise assembly of complex IV

  • Identifying critical checkpoints where disease mutations exert effects

  • Establishing the temporal sequence of assembly factor recruitment

• Tissue-specific pathology insights:

  • Comparing PET100 expression and localization across affected tissues

  • Correlating expression patterns with tissue-specific disease manifestations

  • Identifying compensatory mechanisms in resistant tissues

• Therapeutic target identification:

  • Screening for compounds that restore proper PET100 localization

  • Identifying druggable interaction partners in the assembly pathway

  • Developing biomarkers for treatment response monitoring

• Genetic modifier discovery:

  • Using PET100 as a readout for genetic screens

  • Identifying genes that buffer or exacerbate PET100 deficiency

  • Understanding variable penetrance in mitochondrial disorders

• Mitochondrial quality control mechanisms:

  • Investigating how dysfunctional complex IV assembly triggers mitophagy

  • Exploring connections between PET100 and mitochondrial stress responses

  • Elucidating the relationship between assembly defects and ROS production

Table 2: Potential Contributions of PET100 Research to Mitochondrial Disease Understanding

This framework highlights how PET100 antibody research contributes to a systems-level understanding of mitochondrial disease mechanisms .