At5g08670 Antibody

What is the At5g08670 gene and why are antibodies against it important for plant research?

At5g08670 is a gene in Arabidopsis thaliana that encodes a subunit of the ATP synthase complex, which plays a critical role in energy metabolism within plant mitochondria. Antibodies targeting this protein are essential tools for investigating mitochondrial function, respiratory chain activity, and ATP synthesis in plants. These antibodies enable researchers to monitor protein expression levels, localization patterns, and post-translational modifications under various experimental conditions, including environmental stresses like temperature changes. The importance of these antibodies is highlighted in studies investigating how ATP synthase functions as a respiratory bottleneck during chilling stress .

What are the optimal sample preparation techniques for At5g08670 antibody applications in Western blotting?

For optimal Western blot results with At5g08670 antibodies, mitochondrial isolation is a critical first step. The most effective protocol involves:

• Homogenizing plant tissue (preferably young leaves) in extraction buffer containing 0.3 M sucrose, 25 mM HEPES, 10 mM EDTA, pH 7.5, with freshly added 1 mM DTT and protease inhibitor cocktail

• Filtering through Miracloth followed by differential centrifugation (1,000g, 5,000g, and finally 18,000g)

• Resuspending the mitochondrial pellet in a minimal volume of resuspension buffer

• Protein quantification using Bradford or BCA assay

For the Western blot itself, load 10-20 μg of mitochondrial protein per lane on 12% SDS-PAGE gels, followed by wet transfer to PVDF membranes at 100V for 1 hour. Block with 5% non-fat dry milk in TBS-T and incubate with the primary At5g08670 antibody (1:1000 dilution) overnight at 4°C. Detection using ECL provides cleaner results than colorimetric methods .

How can I verify the specificity of an At5g08670 antibody for my plant species of interest?

Verifying antibody specificity for At5g08670 across different plant species requires several validation steps:

• Positive and negative controls: Include mitochondrial extracts from wild-type Arabidopsis as a positive control and ATP synthase knockdown lines (such as anti-ATP3-3 or anti-ATP3-6) as negative controls

• Peptide competition assay: Pre-incubate the antibody with excess synthetic peptide used for immunization before applying to your Western blot

• Cross-reactivity testing: Test the antibody against mitochondrial extracts from your species of interest alongside Arabidopsis samples

• Mass spectrometry validation: Confirm the identity of the immunoprecipitated protein by LC-MS/MS analysis

The antibody's specificity can be further validated through immunolocalization studies using confocal microscopy, comparing the signal pattern with known mitochondrial markers like MitoTracker. Remember that sequence conservation of ATP synthase subunits varies across plant species, so epitope analysis using sequence alignment tools is recommended before proceeding with non-model species .

How should I design experiments to measure ATP synthase activity using the At5g08670 antibody under different temperature conditions?

To effectively measure ATP synthase activity across temperature conditions while using At5g08670 antibody for protein quantification:

• Experimental design: Implement a factorial design with at least three temperature points (e.g., 4°C, 15°C, and 25°C) and three plant treatments (warm-grown, cold-acclimated, and cold-shocked)

• Activity measurement: Use a coupled enzymatic assay where ATP synthesis is linked to NADH oxidation monitored spectrophotometrically at 340 nm

• Temperature control: Maintain precise temperature control during both mitochondrial isolation and assay procedures using water-jacketed chambers

• Antibody application: Quantify At5g08670 protein levels via Western blot to normalize enzymatic activities to protein abundance

A recommended approach involves measuring both ATP hydrolysis rates and ATP/O ratios at different temperatures. For ATP hydrolysis, use a coupled enzymatic assay where ADP production is linked to NADH oxidation. For ATP/O ratios, simultaneously measure ATP production using the luciferase assay and oxygen consumption with a Clark-type electrode.

Data analysis should include calculating Q10 values (temperature coefficients) to determine the temperature sensitivity of ATP synthase relative to other respiratory components. Statistical analysis should employ two-way ANOVA to assess the interaction between temperature and plant treatment .

What controls should be included when measuring mitochondrial membrane potential alongside At5g08670 antibody labeling?

When measuring mitochondrial membrane potential (ΔΨm) in conjunction with At5g08670 antibody labeling, include these essential controls:

• Positive controls:

  • Fully energized mitochondria (State 2 respiration with substrate but no ADP)

  • Oligomycin A-treated mitochondria (maximal membrane potential)

• Negative controls:

  • FCCP or CCCP-treated mitochondria (complete uncoupling)

  • Heat-denatured mitochondria (background signal)

• Process controls:

  • Titrate potassium cyanide (KCN) to incrementally inhibit cytochrome pathway

  • Add n-propyl gallate (nPG) to inhibit alternative oxidase pathway

For membrane potential measurements, tetramethylrhodamine methyl ester (TMRM) is recommended at 0.5 μM final concentration. Calibrate the fluorescence signal using a K+ gradient with valinomycin. Simultaneous oxygen consumption measurements should be performed using an Oroboros O2K-Fluorescence LED2 system or similar equipment that allows concurrent monitoring of respiration and fluorescence.

For quantification, calculate the relative changes in membrane potential during different respiratory states (State 2, State 3, State 4) and at different temperatures, correlating these measurements with At5g08670 protein abundance determined by immunoblotting .

How can I use the At5g08670 antibody to investigate ATP synthase assembly under stress conditions?

To investigate ATP synthase assembly under stress conditions using At5g08670 antibody:

• Blue-Native PAGE approach:

  • Solubilize mitochondrial membranes with digitonin (6g/g protein)

  • Separate complexes on 3-12% gradient BN-PAGE

  • Perform second dimension SDS-PAGE for subunit analysis

  • Western blot using At5g08670 antibody to detect the target subunit

• Co-immunoprecipitation strategy:

  • Crosslink proteins in intact mitochondria using DSP (dithiobis-succinimidyl propionate)

  • Solubilize with 1% digitonin

  • Immunoprecipitate using At5g08670 antibody coupled to Protein A/G beads

  • Analyze precipitated complexes by mass spectrometry

• Comparative analysis:

  • Compare ATP synthase assembly between normal and stress conditions (e.g., cold, heat, drought)

  • Quantify the ratio of assembled complex to free subunits

For cold stress specifically, expose plants to 4°C treatment with time points at 1, 3, and 5 days, comparing to warm-grown controls. Use selected reaction monitoring (SRM) mass spectrometry to quantify absolute abundance of ATP synthase subunits, correlating these measurements with ATP synthase activity and respiration rates .

How can I use the At5g08670 antibody to investigate the relationship between mitochondrial membrane lipid composition and ATP synthase function?

To investigate the relationship between mitochondrial membrane lipid composition and ATP synthase function using At5g08670 antibody:

• Integrated lipidomics and proteomics approach:

  • Isolate mitochondria using Percoll gradient purification

  • Split the preparation for parallel protein and lipid analyses

  • Extract lipids using a modified Bligh and Dyer method with MTBE

  • Analyze lipids via LC-MS/MS lipidomics

  • Quantify At5g08670 protein abundance via immunoblotting

• Correlation analysis:

  • Create volcano plots of lipid molecular features that change significantly between conditions

  • Perform principal component analysis (PCA) to identify patterns

  • Use hierarchical clustering to group lipid changes

  • Correlate specific lipid changes (especially cardiolipin species) with At5g08670 protein levels

• Functional validation:

  • Reconstitute purified ATP synthase into liposomes with defined lipid compositions

  • Measure ATP synthesis activity in these proteoliposomes

  • Compare activity in different lipid environments

In the analysis, focus particularly on cardiolipin (CL), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) content, as these lipids are known to interact with and influence ATP synthase activity. A comprehensive approach would include extraction ion chromatogram (EIC) analysis of key lipids like CL species with m/z values around 663.45, and correlation of their abundance with ATP synthase activity at different temperatures .

What approaches can be used to study the dynamic interaction between At5g08670 and other ATP synthase subunits during cold acclimation?

To study dynamic interactions between At5g08670 and other ATP synthase subunits during cold acclimation:

• Time-resolved interaction studies:

  • Collect plant material at multiple time points during cold acclimation (6h, 12h, 24h, 72h, 7d)

  • Perform crosslinking of mitochondrial proteins at each time point

  • Use the At5g08670 antibody for immunoprecipitation

  • Analyze co-precipitated proteins by LC-MS/MS

  • Quantify changes in interaction partners over time

• Fluorescence resonance energy transfer (FRET) analysis:

  • Create fusion proteins with At5g08670 and other subunits tagged with appropriate fluorophores

  • Measure FRET efficiency in isolated mitochondria under different temperature conditions

  • Calculate interaction distances based on FRET measurements

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Expose purified ATP synthase complexes to D2O under different temperature conditions

  • Analyze the rate and extent of hydrogen-deuterium exchange

  • Map temperature-dependent conformational changes involving At5g08670

For data analysis, implement statistical methods for time-series data, including repeated measures ANOVA and non-linear regression modeling to characterize the kinetics of complex assembly during cold acclimation. Compare these kinetics with the development of cold tolerance phenotypes to establish physiological relevance .

How can At5g08670 antibody be used to investigate the relationship between ATP synthase activity and alternative respiratory pathways in plants?

To investigate the relationship between ATP synthase and alternative respiratory pathways using At5g08670 antibody:

• Respiratory flux analysis:

  • Isolate mitochondria from wild-type and ATP synthase-modified plants

  • Measure oxygen consumption rates in different respiratory states

  • Use specific inhibitors to distinguish pathways:

    • KCN for cytochrome pathway inhibition

    • SHAM or nPG for alternative oxidase (AOX) inhibition

    • Oligomycin for ATP synthase inhibition

  • Quantify At5g08670 and AOX protein levels via immunoblotting

• Membrane potential correlation:

  • Simultaneously measure oxygen consumption and membrane potential

  • Compare ΔΨm during State 3 respiration versus alternative pathway respiration

  • Correlate membrane potential differences with At5g08670 and AOX protein abundance

• In vivo respiratory analysis:

  • Use stable isotope labeling to track carbon flux through different respiratory pathways

  • Correlate respiratory partitioning with ATP synthase activity

  • Compare wild-type plants with ATP synthase knockdown lines (anti-ATP3-3, anti-ATP3-6)

Research data should be presented as a matrix of respiratory parameters (State 2, State 3, State 4, uncoupled, and alternative pathway respiration rates) measured at different temperatures (4°C, 15°C, 25°C) across different plant treatments (warm-grown, cold-acclimated, cold-shocked). Statistical analysis should include correlation tests between At5g08670 protein levels and the capacity of alternative pathways, particularly under stress conditions .

How should I interpret contradictory results between At5g08670 antibody detection and ATP synthase activity measurements?

When faced with contradictions between At5g08670 antibody detection and ATP synthase activity measurements:

• Potential causes of discrepancy:

  • Post-translational modifications affecting enzyme activity but not antibody detection

  • Assembly issues where the subunit is present but not incorporated into functional complexes

  • Temperature-dependent conformational changes affecting epitope accessibility

  • Differential stability of the ATP synthase complex under experimental conditions

• Systematic investigation approach:

  • Compare ATP hydrolysis vs. ATP synthesis activities (they may be differentially affected)

  • Analyze ATP/O ratios at different temperatures to identify coupling efficiency issues

  • Examine respiratory control ratios (RCRs) and uncoupling control ratios (UCRs)

  • Use Blue-Native PAGE to assess complex integrity alongside Western blotting

• Critical data interpretation:

  • Consider temperature coefficients (Q10) for different aspects of ATP synthase function

  • Evaluate whether ATP synthase activity correlates better with specific subunit abundances

  • Compare results with known mutants or knockdown lines (e.g., anti-ATP3-3, anti-ATP3-6)

When analyzing discrepancies, remember that respiratory bottlenecks may occur at different points depending on temperature. At low temperatures (4°C), ATP synthase often becomes rate-limiting despite adequate subunit abundance, as demonstrated by membrane potential measurements showing higher proton motive force but lower ATP synthesis rates compared to measurements at 25°C .

What are the potential pitfalls in quantifying At5g08670 protein levels in plant mitochondrial samples?

Researchers should be aware of these potential pitfalls when quantifying At5g08670 protein levels:

• Sample preparation challenges:

  • Mitochondrial purity variations affecting quantification

  • Differential extraction efficiency at different temperatures

  • Membrane protein solubilization inconsistencies

  • Proteolytic degradation during isolation procedures

• Technical quantification issues:

  • Non-linear relationship between signal intensity and protein abundance

  • Variations in transfer efficiency during Western blotting

  • Batch-to-batch antibody variability

  • Loading control selection and normalization strategy

• Experimental design considerations:

  • Developmental stage affecting mitochondrial yield and composition

  • Circadian effects on protein expression

  • Light conditions during plant growth affecting mitochondrial properties

  • Soil vs. hydroponic growth systems yielding different results

To address these issues, implement these solutions:

• Use multiple isolation biological replicates (minimum n=4)

• Apply appropriate normalization strategies (total protein, specific mitochondrial markers)

• Consider selected reaction monitoring (SRM) mass spectrometry as an antibody-independent quantification method

• Include internal standards and standard curves for absolute quantification

• Verify results using complementary approaches (e.g., enzymatic activity, RNA expression)

How can I distinguish between direct effects on At5g08670/ATP synthase and secondary metabolic adaptations in temperature response experiments?

To distinguish between direct effects on ATP synthase and secondary metabolic adaptations:

• Temporal resolution approach:

  • Implement high-resolution time-course experiments (minutes to days)

  • Compare immediate responses (direct effects) with longer-term changes (adaptive responses)

  • Track At5g08670 protein abundance, ATP synthase activity, and respiratory parameters in parallel

  • Correlate changes with transcriptional responses of related genes

• Genetic approach:

  • Use ATP synthase knockdown lines (e.g., anti-ATP3-3, anti-ATP3-6)

  • Compare their temperature response with wild-type plants

  • Analyze natural variation in ecotypes with different ATP synthase temperature responses (e.g., Col-0 vs. T1110)

  • Create complementation lines to verify specific subunit effects

• Biochemical discrimination:

  • Measure ATP synthase activity in isolated mitochondria vs. whole tissues

  • Use selective inhibitors to distinguish ATP synthase effects from other processes

  • Implement metabolomic profiling to identify secondary adaptive responses

  • Apply flux analysis using stable isotopes to track metabolic adjustments

The most definitive approach involves combining in vitro biochemical assays (using purified mitochondria with defined substrates) with in vivo physiological measurements. This allows researchers to distinguish direct temperature effects on ATP synthase properties from plant-level adaptive responses that might compensate for these effects through alternative pathways or metabolic remodeling .

How can the At5g08670 antibody be used in studies investigating the relationship between mitochondrial dynamics and energy production under stress?

The At5g08670 antibody can be instrumental in exploring mitochondrial dynamics and energy production through:

• Combined microscopy and biochemical approach:

  • Perform immunogold labeling with At5g08670 antibody for transmission electron microscopy

  • Correlate ATP synthase distribution with mitochondrial morphology changes during stress

  • Use super-resolution microscopy (STORM/PALM) for nanoscale organization studies

  • Combine with live-cell imaging using mitochondrial morphology markers

• Mitochondrial isolation from distinct populations:

  • Use density gradient centrifugation to separate mitochondrial subpopulations

  • Quantify At5g08670 distribution across different mitochondrial fractions

  • Correlate ATP synthase abundance with functional parameters in each subpopulation

  • Track changes in distribution patterns during stress responses

• Integration with mitochondrial dynamics machinery:

  • Investigate co-localization of At5g08670 with fusion/fission proteins

  • Analyze how disruption of mitochondrial dynamics affects ATP synthase distribution

  • Examine the relationship between cristae remodeling and ATP synthase organization

  • Study mitochondria-associated membranes (MAMs) and their role in ATP synthase regulation

This research direction provides insights into how plants compartmentalize bioenergetic functions during stress adaptation. Focus on analyzing how mitochondrial fusion, fission, and mobility correlate with ATP synthase clustering and activity, particularly during temperature transitions where membrane properties and energy demands change significantly .

What role might post-translational modifications of At5g08670 play in regulating ATP synthase activity during environmental stress responses?

Post-translational modifications (PTMs) of At5g08670 likely play crucial roles in ATP synthase regulation:

• Comprehensive PTM mapping approach:

  • Immunoprecipitate ATP synthase complex using At5g08670 antibody

  • Analyze by LC-MS/MS with PTM-specific enrichment strategies:

    • Phosphopeptide enrichment using TiO2 or IMAC

    • Redox modification analysis using differential alkylation

    • Lysine modification detection (acetylation, ubiquitination, sumoylation)

  • Compare PTM profiles between normal and stress conditions

• Functional validation of PTMs:

  • Generate site-specific mutants mimicking or preventing specific modifications

  • Express modified variants in ATP synthase knockdown backgrounds

  • Measure the impact on ATP synthase activity, assembly, and stability

  • Correlate with physiological responses to environmental stresses

• Regulatory enzyme identification:

  • Use proximity labeling approaches with At5g08670 as bait

  • Identify kinases, phosphatases, and other modifying enzymes associated with ATP synthase

  • Validate these interactions through co-immunoprecipitation with At5g08670 antibody

  • Test the effects of inhibitors targeting these regulatory enzymes

Current research suggests that phosphorylation of ATP synthase subunits increases during cold stress, potentially as a mechanism to fine-tune activity when reduced temperatures would otherwise limit function. Redox modifications may also become more prominent during temperature stress as a response to altered ROS production. Using the At5g08670 antibody to track these modifications provides a direct link between PTM status and functional outcomes .

How can At5g08670 antibody be used in comparative studies across plant species with different temperature adaptations?

To use At5g08670 antibody in comparative studies across plant species with different temperature adaptations:

• Cross-species applicability assessment:

  • Perform sequence alignment of At5g08670 homologs across target species

  • Identify conserved epitope regions recognized by the antibody

  • Test cross-reactivity through Western blotting of mitochondrial extracts

  • Optimize immunodetection conditions for each species

• Comparative physiological framework:

  • Select plant species with contrasting temperature adaptations:

    • Cold-adapted (e.g., alpine or boreal species)

    • Heat-adapted (e.g., desert or tropical species)

    • Temperate species with wide temperature tolerance

  • Standardize growth and stress treatment protocols across species

  • Measure At5g08670 homolog abundance, ATP synthase activity, and respiratory parameters

• Evolutionary adaptation analysis:

  • Correlate ATP synthase properties with species' native temperature ranges

  • Examine sequence variations in ATP synthase subunits across species

  • Relate structural differences to thermal stability and activity profiles

  • Test chimeric proteins combining subunits from different species to identify key adaptation domains

This approach allows researchers to uncover evolutionary strategies for maintaining bioenergetic efficiency across temperature ranges. Special attention should be given to the ATP synthase catalytic site, rotor components, and membrane-anchoring domains, as these regions often show adaptive variations in thermally diverse organisms. When interpreting comparative data, consider that absolute antibody signal intensity may not be directly comparable across species, necessitating complementary quantification methods .