At5g13450 Antibody

What is AT5G13450 and what role does it play in plant mitochondria?

AT5G13450 encodes ATP5, a critical subunit of mitochondrial F0F1-ATP synthase in Arabidopsis. This complex (also called Complex V) synthesizes ATP from ADP and inorganic phosphate (Pi) using the proton motive force created by respiratory electron transport . ATP5 is essential for energy production in plant cells, participating in the coupling mechanism that converts the electrochemical proton gradient into chemical energy. The protein is localized to the mitochondrial membrane, where it functions as part of the ATP synthase machinery responsible for oxidative phosphorylation .

What types of ATP5 antibodies are available for plant research?

Commercial antibodies targeting ATP5 include monoclonal and polyclonal options. PhytoAB offers two specific antibodies: PHY0590S and PHY1132S, with the former utilizing a KLH-conjugated synthetic peptide of ATP5 derived from Arabidopsis thaliana AT5G13450 as the immunogen . When selecting an antibody, researchers should consider:

• Antibody type (monoclonal vs. polyclonal)

• Host species (to avoid cross-reactivity in co-immunostaining)

• Epitope location (important for detecting specific protein domains)

• Validation data in plant tissues (particularly cross-reactivity with related ATP synthase subunits)

• Species cross-reactivity (if working with plants other than Arabidopsis)

What are the recommended protocols for validating AT5G13450 antibody specificity?

Before using an ATP5 antibody in crucial experiments, validation is essential:

• Western blot analysis using:

  • Positive control: mitochondria-enriched fractions from wild-type plants

  • Negative control: ATP5 knockdown lines (if available)

  • Molecular weight verification: ATP5 should appear at the expected size

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Run parallel Western blots with treated and untreated antibody

  • Signal disappearance confirms specificity

• Immunoprecipitation followed by mass spectrometry:

  • Confirms antibody captures the intended protein

  • Identifies potential cross-reacting proteins

• Comparison with multiple antibodies:

  • Use antibodies recognizing different epitopes of ATP5

  • Consistent results increase confidence in specificity

How can I optimize Western blot protocols for reliable detection of ATP5 in plant mitochondria?

Optimizing Western blot protocols for ATP5 detection requires special considerations for membrane proteins:

When troubleshooting, cold-induced changes in ATP5 can affect detection sensitivity, as observed in studies examining ATP synthase activity at varying temperatures .

What considerations are important when using ATP5 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (co-IP) with ATP5 antibodies presents unique challenges due to the protein's membrane localization and complex formation:

• Sample preparation optimization:

  • Use gentle detergents (0.5-1% digitonin or 1% n-dodecyl β-D-maltoside)

  • Include physiological salt concentrations (150-250 mM NaCl)

  • Maintain neutral pH (7.2-7.6)

  • Add protease inhibitor cocktail and phosphatase inhibitors

• Pre-clearing strategy:

  • Pre-clear lysates with Protein A/G beads

  • Include control IgG from the same species as the ATP5 antibody

  • Remove mitochondrial debris by centrifugation (10,000g for 10 min)

• Antibody coupling approaches:

  • Direct coupling: Add ATP5 antibody to pre-cleared lysate

  • Bead coupling: Pre-couple antibody to Protein A/G beads

  • Cross-linking: Consider cross-linking antibody to beads to prevent co-elution

• Washing conditions:

  • Step gradient of salt concentrations (150 mM to 300 mM NaCl)

  • Include 0.1% detergent in wash buffers

  • Perform 4-5 washes to minimize background

• Elution methods:

  • Denaturing: SDS sample buffer at 70°C (not boiling)

  • Native: Competitive elution with excess immunizing peptide

The effectiveness of these methods has been demonstrated in studies examining ATP synthase subunit interactions under various temperature conditions .

How should I design experiments to investigate ATP5 activity and abundance during cold stress response?

Based on detailed mitochondrial studies, experimental design for cold stress response should consider:

• Treatment design:

  • Cold shock (CS): Direct exposure to 4°C for 24-48 hours

  • Cold acclimation (CA): Gradual temperature decrease over 5-7 days

  • Warm grown (WG) controls: Maintained at 21-25°C

  • Recovery: Return to normal temperature after cold exposure

• Sampling strategy:

  • Time-course collection (0h, 6h, 12h, 24h, 72h, 7d)

  • Separate analysis of shoots and roots

  • Collection at consistent time of day to control for circadian effects

  • Flash freezing of samples in liquid nitrogen

• Analytical approaches:

  • Western blotting with ATP5 antibodies for protein abundance

  • RT-qPCR for transcript levels

  • Respiratory measurements:

    • Oxygen consumption rates in different states

    • ATP production rates

    • Membrane potential measurements using TMRM

  • ATP synthase activity assays at different temperatures

• Key parameters to measure:

Research has shown that ATP synthase activity is inhibited to a greater extent than electron transport at low temperatures, potentially creating a respiratory bottleneck .

How can I effectively measure ATP synthase activity at different temperatures to study cold adaptation?

Measuring ATP synthase activity across temperature ranges requires methodological precision:

• Isolation of intact mitochondria:

  • Use density gradient centrifugation for high purity

  • Verify integrity using respiratory control ratios (RCR > 3)

  • Maintain consistent protein concentration (0.2-0.5 mg/ml)

• ATP synthase activity measurement options:

  • Coupled enzymatic assay: Measures ATP hydrolysis through coupled reactions

    • Advantage: High sensitivity

    • Challenge: Temperature affects coupled enzymes

  • Direct measurement: ATP production using luciferase

    • Advantage: Direct physiological relevance

    • Challenge: Requires rapid sample processing

• Temperature control considerations:

  • Pre-equilibrate all reagents to measurement temperature

  • Use water-jacketed chambers with circulating temperature-controlled water

  • Include temperature probe in reaction chamber

  • Allow sufficient equilibration time (5-10 minutes)

• Data analysis approach:

  • Calculate Q10 values (activity ratio between temperatures 10°C apart)

  • Determine Arrhenius activation energies

  • Compare with electron transport chain activity Q10

  • Identify rate-limiting steps using inhibitor titrations

Research demonstrates that ATP synthase activity shows a more pronounced decline at 4°C than electron transport chain activity, with Q10 values of approximately 2.5-3.0 for ATP synthase compared to 1.8-2.2 for electron transport .

What are the best approaches for simultaneous measurement of oxygen consumption and membrane potential?

Based on advanced methodologies used in mitochondrial research:

• Equipment setup:

  • Oroboros O2K-Fluorescence LED2 system or equivalent

  • Clark-type oxygen electrode with fluorescence detection capability

  • Temperature-controlled reaction chamber

  • Data acquisition software

• Probe selection:

  • Oxygen: Clark electrode or optical sensor

  • Membrane potential: TMRM (tetramethylrhodamine methyl ester) or Safranin O

    • TMRM concentration: 0.5-1.0 μM (non-quenching mode)

    • Excitation/emission: 548/574 nm for TMRM

• Experimental sequence:

  • Calibrate oxygen electrode at measurement temperature

  • Add mitochondria (0.2-0.5 mg/ml) to respiration medium

  • Record baseline (State 2) respiration and membrane potential

  • Add ADP (250-500 μM) to initiate State 3 respiration

  • Monitor transition to State 4 after ADP depletion

  • Add uncoupler (FCCP, 0.5-1.0 μM) to collapse membrane potential

  • Add inhibitors (KCN, oligomycin) to assess contributions of pathways

• Data interpretation:

Studies show that membrane potential in State 3 decreases more dramatically at 4°C than at 25°C, supporting the hypothesis that ATP synthase becomes more limiting at low temperatures .

What statistical approaches should I use when analyzing ATP5 abundance data from different temperature treatments?

For robust statistical analysis of ATP5 abundance data:

• Experimental design considerations:

  • Minimum of 3-4 biological replicates

  • 2-3 technical replicates per biological sample

  • Include time-matched controls

  • Account for batch effects in experimental design

• Normalization strategies:

  • Reference proteins: VDAC, HSP60, or other stable mitochondrial proteins

  • Total protein normalization (Ponceau S or SYPRO Ruby staining)

  • Consider multiple normalization methods and compare results

• Statistical testing approach:

  • For normally distributed data: ANOVA with post-hoc tests (Tukey's HSD)

  • For non-parametric data: Kruskal-Wallis with Dunn's post-hoc test

  • For time-course experiments: Repeated measures ANOVA

  • For complex designs: Linear mixed-effects models accounting for random effects

• Advanced analytical considerations:

  • Principal component analysis for multivariate data

  • Hierarchical clustering for pattern identification

  • Correlation analysis between protein abundance and physiological parameters

Researchers have employed these approaches when analyzing significant changes in ATP synthase subunit abundance during cold acclimation, particularly when examining correlations between protein levels and respiratory parameters like ATP/O ratios .

How do I interpret contradictory results between ATP5 protein abundance and ATP synthase activity?

When faced with discrepancies between protein levels and enzyme activity:

• Potential mechanistic explanations:

  • Post-translational modifications affecting enzyme activity

  • Alterations in complex assembly despite stable subunit levels

  • Changes in lipid environment affecting enzyme function

  • Temperature-dependent conformational changes

  • Substrate availability limitations

• Verification approaches:

  • Blue Native PAGE to assess complex assembly

  • Phospho-specific antibodies to detect regulatory modifications

  • Lipid analysis of mitochondrial membranes

  • In-gel activity assays for assembled complexes

  • Detailed enzyme kinetics at different temperatures

• Case study from research data:
  ATP synthase activity may decrease dramatically at 4°C despite minimal changes in ATP5 protein abundance, suggesting that temperature directly affects enzyme function rather than protein levels . This is supported by:

  Parameter	25°C	4°C	Fold Change
  ATP5 protein abundance	Baseline	0.8-1.0x	Minimal change
  ATP synthase activity	Baseline	0.2-0.3x	3-5 fold decrease
  State 3 respiration	Baseline	0.3-0.4x	2.5-3 fold decrease
  Uncoupled respiration	Baseline	0.5-0.6x	~2 fold decrease

  This pattern indicates that temperature affects catalytic efficiency more than protein stability .

What factors could explain variable ATP5 antibody performance between experiments?

When troubleshooting inconsistent ATP5 antibody performance:

• Antibody-related factors:

  • Lot-to-lot variability in commercial antibodies

  • Antibody degradation from improper storage or freeze-thaw cycles

  • Epitope accessibility changes under different sample preparation methods

  • Cross-reactivity with homologous ATP synthase subunits

• Sample-related factors:

  • Plant growth conditions affecting post-translational modifications

  • Tissue-specific isoform expression

  • Developmental stage variations

  • Oxidation of proteins during sample preparation

  • Protein degradation during extraction

• Technical factors:

  • Buffer composition effects on epitope exposure

  • Temperature effects during incubation steps

  • Variations in transfer efficiency during Western blotting

  • Detection system sensitivity fluctuations

• Validation and optimization approach:

  • Always include positive control samples

  • Test new antibody lots against reference samples

  • Optimize fixation conditions for immunohistochemistry

  • Consider native vs. denaturing conditions

  • Test multiple antibody concentrations in a dot-blot format

Research on ATP synthase has shown that protein extraction methods can significantly impact antibody recognition, particularly for membrane-associated proteins like ATP5 .

How can I determine if changes in ATP5 are causative or adaptive in cold stress response?

Distinguishing between causative roles and adaptive responses requires sophisticated experimental approaches:

• Genetic manipulation strategies:

  • RNAi or CRISPR knockdown of ATP5

  • Inducible expression systems (e.g., dexamethasone-inducible)

  • Comparison of ATP5 knockdown effects under normal and stress conditions

  • Introduction of temperature-insensitive ATP synthase variants

• Time-course experimental design:

  • High-resolution sampling during early cold response

  • Correlation of ATP5 changes with physiological parameters

  • Recovery experiments after cold stress

  • Comparison of rapid vs. gradual temperature changes

• Systems biology approaches:

  • Metabolomic analysis to identify downstream effects

  • Transcriptomic profiling for coordinated gene expression

  • Flux analysis to determine metabolic bottlenecks

  • Network analysis to position ATP5 in stress response pathways

• Natural variation studies:

  • Compare Arabidopsis ecotypes with differential cold tolerance

  • Screen for natural variants in ATP5 sequence or regulation

  • Correlate ATP5 sequence polymorphisms with functional differences

Research has employed ATP synthase knockdown lines with dexamethasone-inducible atp3-3 and atp5 constructs to demonstrate that ATP synthase limitation is an adaptive response rather than merely a physical constraint, as plants with reduced ATP synthase show improved low-temperature respiration profiles .

What techniques can distinguish between changes in ATP5 abundance versus ATP synthase complex assembly?

Distinguishing between individual subunit changes and complex assembly requires specialized approaches:

• Blue Native PAGE techniques:

  • Gentle solubilization using digitonin (4-8 g/g protein)

  • Gradient gels (3-12% or 4-16%) for optimal separation

  • In-gel activity staining for ATP synthase

  • Second dimension SDS-PAGE for subunit composition

• Sucrose gradient ultracentrifugation:

  • Separation of assembled complexes by size

  • Western blot analysis of gradient fractions

  • Comparison of ATP5 distribution across fractions

  • Activity measurements in gradient fractions

• Cross-linking mass spectrometry:

  • Chemical cross-linking of protein complexes

  • Digestion and mass spectrometric analysis

  • Identification of subunit interaction partners

  • Quantitative comparison between conditions

• Fluorescence microscopy approaches:

  • FRET between labeled subunits to assess proximity

  • Superresolution imaging of complex distribution

  • Colocalization analysis with multiple labeled subunits

  • Live-cell imaging during temperature transitions

Research on ATP synthase has employed selected reaction monitoring (SRM) to quantify the abundance of specific ATP synthase subunits and correlate subunit stoichiometry with complex function under different temperature conditions .

What emerging technologies might advance ATP5 research in plant stress physiology?

Cutting-edge approaches for ATP5 research include:

• Cryo-electron microscopy:

  • High-resolution structures of plant ATP synthase

  • Visualization of temperature-dependent conformational changes

  • Comparison of ATP synthase from cold-adapted vs. temperate plants

  • Structural basis for regulatory interactions

• Single-molecule techniques:

  • Monitoring ATP synthase rotational catalysis in real-time

  • Temperature-dependent kinetic analysis

  • Direct measurement of proton pumping

  • Force generation measurements at different temperatures

• Genome editing applications:

  • Precise modification of ATP5 regulatory sites

  • Introduction of temperature-insensitive variants

  • Creation of tagged variants for in vivo tracking

  • Modulation of ATP synthase oligomerization

• Biophysical approaches:

  • Nanoscale thermometry of mitochondrial microenvironments

  • High-resolution respirometry with simultaneous parameter monitoring

  • Membrane fluidity measurements correlating with ATP synthase function

  • Real-time ATP imaging in living plant cells

These approaches could help resolve the mechanistic basis for ATP synthase inhibition at low temperatures, potentially leading to crops with improved cold tolerance through optimized energy metabolism .

How might comparative studies across plant species inform our understanding of ATP5 function?

Comparative studies offer valuable insights:

• Evolutionary perspectives:

  • Comparison of ATP5 sequence and structure across plant phylogeny

  • Identification of conserved vs. variable domains

  • Correlation of sequence features with thermal habitat

  • Reconstruction of ancestral ATP5 sequences

• Cross-species functional analysis:

  • ATP synthase activity comparison from plants with different thermal optima

  • Temperature-dependent kinetics across species

  • Heterologous expression of ATP5 variants

  • Chimeric constructs to identify temperature-sensitive domains

• Adaptive traits in extremophiles:

  • Study of ATP5 from cold-adapted plants (e.g., Antarctic species)

  • Comparison with heat-tolerant species

  • Identification of compensatory mechanisms

  • Potential for introducing beneficial traits into crops

• Method development considerations:

  • Optimization of ATP5 antibodies for cross-species recognition

  • Standardized assays for comparative analyses

  • Bioinformatic pipelines for sequence-function correlation

  • Physiological relevance of in vitro measurements

Comparing different Arabidopsis ecotypes, researchers identified natural variation in ATP synthase function at low temperatures, with certain ecotypes (like T1110) showing improved respiratory performance at 4°C compared to Col-0, suggesting genetic adaptations in ATP synthase that could be exploited for crop improvement .