At5g08690 Antibody

What is AT5G08690 and why is it significant in plant mitochondrial research?

AT5G08690 encodes a beta-2 subunit of mitochondrial ATP synthase (also called Complex V) in Arabidopsis thaliana. This protein is critical for ATP synthesis from ADP and inorganic phosphate (Pi) using the proton motive force generated by respiratory electron transport. The significance of this protein lies in its essential role in cellular energy production, making it a crucial target for studying plant bioenergetics, stress responses, and metabolic regulation . Antibodies against AT5G08690 enable researchers to detect, quantify, and localize this protein within experimental systems, providing insights into mitochondrial function under various conditions. The study of ATP synthase components has significantly advanced our understanding of energy conservation mechanisms in plants and how they adapt to environmental stresses.

How does AT5G08690 antibody differ from antibodies targeting other ATP synthase subunits?

AT5G08690 antibody specifically targets the beta-2 subunit of mitochondrial ATP synthase, which distinguishes it from antibodies against other ATP synthase components such as alpha subunits or gamma subunits. The specificity of this antibody is determined by its immunogen sequence, which has been designed to recognize the unique epitopes of AT5G08690 (P83484) . Importantly, the sequence used for immunization shows 100% homology with AT5G08670 and AT5G08680, which are paralogous ATP synthase beta subunits in Arabidopsis thaliana . This cross-reactivity with closely related paralogs must be considered when interpreting experimental results. Unlike antibodies targeting ATP synthase components in the F₀ portion (membrane-embedded), the AT5G08690 antibody detects components of the F₁ portion (matrix-facing), which has implications for experimental design, particularly in fractionation studies or when membrane integrity is a consideration.

What is the cross-reactivity profile of AT5G08690 antibody across different plant species?

The AT5G08690 antibody demonstrates broad cross-reactivity across multiple plant species due to the high conservation of ATP synthase components. Based on sequence homology analysis, the antibody has confirmed reactivity with proteins from numerous species including:

This extensive cross-reactivity makes the antibody particularly valuable for comparative studies across different plant taxonomic groups . When using this antibody with a non-validated species, researchers should first perform appropriate validation experiments, including western blot analysis with positive controls, to confirm specificity.

What are the optimal protocols for using AT5G08690 antibody in Western blot analysis of plant mitochondrial samples?

For optimal Western blot results with AT5G08690 antibody in plant mitochondrial samples, the following methodological approach is recommended:

• Sample Preparation:

  • Isolate mitochondria using differential centrifugation with a 0.3M sucrose-based isolation buffer (containing 25mM MOPS, 1mM EGTA, and 0.1% BSA)

  • Add protease inhibitors (e.g., PMSF or commercial cocktail) immediately after tissue homogenization

  • Solubilize mitochondrial proteins in sample buffer containing 2% SDS and 50mM DTT

  • Heat samples at 95°C for 5 minutes to denature proteins

• Gel Electrophoresis and Transfer:

  • Separate proteins on 10-12% SDS-PAGE gels

  • Load 5-15μg of mitochondrial protein per lane

  • Transfer to PVDF membrane at 100V for 60 minutes in Tris-glycine buffer with 20% methanol

• Antibody Incubation:

  • Block membrane with 5% non-fat dry milk in TBS-T for 1 hour

  • Incubate with primary AT5G08690 antibody at 1:1000 to 1:5000 dilution overnight at 4°C

  • Wash 3 times with TBS-T (10 minutes each)

  • Incubate with HRP-conjugated secondary antibody (1:10,000 dilution) for 1 hour

  • Visualize using ECL detection

This protocol has been validated to detect ATP synthase beta subunits effectively, with expected band size of approximately 55-60 kDa. For quantitative analysis, include ATP synthase gamma subunit or porin as loading controls.

How can AT5G08690 antibody be effectively used in immunoprecipitation studies to investigate protein-protein interactions in ATP synthase complexes?

For effective immunoprecipitation of ATP synthase complexes using AT5G08690 antibody:

• Pre-clearing Step:

  • Prepare mitochondrial lysate in non-denaturing buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, protease inhibitors)

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding

• Antibody Binding:

  • Incubate 2-5μg of AT5G08690 antibody with 500μg of pre-cleared lysate overnight at 4°C with gentle rotation

  • Add pre-washed Protein A/G beads and incubate for additional 3-4 hours

• Washing and Elution:

  • Wash beads 4-5 times with washing buffer (same as lysis buffer but with 0.1% NP-40)

  • Elute protein complexes with 0.1M glycine (pH 2.5) or SDS sample buffer

• Analysis:

  • Analyze immunoprecipitated complexes by SDS-PAGE followed by western blotting, silver staining, or mass spectrometry

This approach is particularly useful for identifying novel interaction partners of ATP synthase beta subunits and investigating how these interactions may change under different stress conditions, such as cold acclimation or heat stress as observed in Kerbler's mitochondrial studies .

What considerations are important when using AT5G08690 antibody for immunohistochemistry in plant tissue sections?

When using AT5G08690 antibody for immunohistochemistry in plant tissues, several critical factors must be considered:

• Tissue Fixation and Processing:

  • Use freshly prepared 4% paraformaldehyde in PBS for fixation (12-24 hours)

  • Carefully dehydrate tissues through ethanol series (30-100%)

  • Embed in paraffin or LR White resin depending on the required resolution

  • Cut thin sections (5-10μm) using a rotary microtome

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval (10mM citrate buffer, pH 6.0) if using paraffin sections

  • For resin sections, etching with saturated sodium metaperiodate may improve antibody access

• Antibody Dilution and Incubation:

  • Optimal dilution typically ranges from 1:50 to 1:200

  • Incubate sections with primary antibody in humid chamber overnight at 4°C

  • Use fluorescent or enzyme-conjugated secondary antibodies for detection

• Controls and Validation:

  • Include negative controls (omitting primary antibody)

  • Use tissues from ATP synthase knockdown lines as specificity controls

  • Consider dual labeling with mitochondrial markers (e.g., porin antibodies) to confirm mitochondrial localization

• Signal Interpretation:

  • The ATP synthase beta subunit should show punctate staining corresponding to mitochondrial distribution

  • Analysis should account for different tissue types, as mitochondrial density varies between cells

This approach allows visualization of ATP synthase distribution within different plant tissues and cell types, providing insights into tissue-specific energy demands and mitochondrial abundance.

How can AT5G08690 antibody be used to investigate changes in ATP synthase abundance during cold acclimation in plants?

AT5G08690 antibody can serve as a powerful tool for investigating ATP synthase dynamics during cold acclimation through a multi-methodological approach:

• Quantitative Western Blotting:

  • Isolate mitochondria from warm-grown (WG), cold-acclimated (CA), and cold-shocked (CS) plant tissues

  • Perform western blotting with AT5G08690 antibody to quantify ATP synthase beta subunit abundance

  • Normalize against suitable loading controls (porin or TOM40)

  • Use image analysis software for densitometric quantification

• Selective Reaction Monitoring (SRM):

  • Design SRM assays targeting specific peptides unique to AT5G08690

  • Use triple quadrupole mass spectrometry for selective detection of target peptides

  • Compare peptide abundance between treatment conditions using stable isotope-labeled standards

  • This approach provides absolute quantification of protein abundance changes

• Correlation with Functional Parameters:

  • Measure ATP/O ratios in isolated mitochondria using an oxygen electrode

  • Determine membrane potential using fluorescent probes (e.g., safranin O)

  • Correlate ATP synthase abundance with these functional parameters

Research by Kerbler has shown significant changes in mitochondrial energy coupling during cold acclimation, with corresponding alterations in ATP synthase abundance and function . AT5G08690 antibody enables direct monitoring of these changes at the protein level, providing mechanistic insights into plant cold adaptation strategies.

What approaches can be used to study the role of AT5G08690 in maintaining mitochondrial membrane potential (Δψm) under different stress conditions?

To investigate AT5G08690's role in maintaining mitochondrial membrane potential under stress conditions:

• Combined Fluorescence and Respiratory Measurements:

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

  • Use an Oroboros O2K-Fluorescence LED2 system for simultaneous measurement of:

    • Oxygen consumption (respiratory activity)

    • Membrane potential (using safranin O fluorescence)

  • Record changes in Δψm during different respiratory states (State 2, 3, and 4)

• Temperature-Dependent Analysis:

  • Compare Δψm at different temperatures (e.g., 4°C vs. 25°C)

  • Assess the impact of ATP synthase inhibitors (oligomycin A) on Δψm

  • Examine how uncouplers (FCCP) affect Δψm differently in stressed vs. control samples

• Antibody-Based Detection of ATP Synthase Assembly:

  • Use AT5G08690 antibody to assess ATP synthase integrity via BN-PAGE

  • Compare complex assembly/disassembly under different stress conditions

  • Correlate with membrane potential measurements

Data from temperature stress studies have shown that changes in Δψm during State 3 respiration (ADP-stimulated) differ significantly between optimal and stress temperatures, suggesting altered ATP synthase function . Using AT5G08690 antibody allows researchers to connect these functional changes with alterations in protein abundance or complex assembly.

How can AT5G08690 antibody be utilized in proteomics workflows to identify post-translational modifications of ATP synthase under stress conditions?

AT5G08690 antibody can be integrated into advanced proteomics workflows for comprehensive analysis of ATP synthase post-translational modifications:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Use AT5G08690 antibody to immunoprecipitate ATP synthase complexes

  • Digest purified complexes with trypsin

  • Analyze peptides using LC-MS/MS

  • Search for modifications including phosphorylation, acetylation, and oxidation

• Targeted Multiple Reaction Monitoring (MRM):

  • Design MRM assays for known modification sites

  • Use stable isotope-labeled peptide standards for quantification

  • Compare modification levels between control and stress conditions

• Modification-Specific Sample Preparation:

  • For phosphorylation: Enrich phosphopeptides using TiO2 or IMAC

  • For redox modifications: Use differential alkylation strategies

  • For acetylation: Employ anti-acetyllysine antibody enrichment

Table: Common Post-Translational Modifications of ATP Synthase β Subunit

This approach has revealed that ATP synthase subunits undergo significant post-translational modifications during environmental stress, potentially as regulatory mechanisms to adjust energy production to stress conditions .

What are the common challenges when using AT5G08690 antibody in plant samples with high phenolic or secondary metabolite content?

Working with AT5G08690 antibody in plant samples rich in phenolics and secondary metabolites presents several challenges that require specific methodological adaptations:

• Sample Preparation Optimization:

  • Include polyvinylpyrrolidone (PVP, 1-2% w/v) in extraction buffers to bind phenolics

  • Add higher concentrations of reducing agents (5-10 mM DTT) to prevent oxidation

  • Incorporate PVPP (insoluble PVP) during tissue homogenization to adsorb interfering compounds

  • Consider using alternative extraction methods like phenol extraction or TCA/acetone precipitation

• Western Blot Adjustments:

  • Increase washing steps (5-6 washes instead of standard 3)

  • Use higher concentrations of Tween-20 (0.1-0.2%) in wash buffers

  • Add 0.1% SDS to antibody dilution buffer to reduce non-specific binding

  • Consider longer blocking times (2-3 hours) with 5% BSA instead of milk

• Antibody Titration:

  • Perform careful antibody titration to determine optimal concentration

  • Test dilution series from 1:500 to 1:5000 to identify the best signal-to-noise ratio

  • Consider using higher secondary antibody dilutions (1:20,000) to reduce background

Tissues like potato tubers, grape berries, and certain medicinal plants contain high levels of interfering compounds that can mask epitope recognition or create high background. These modifications to standard protocols have been shown to improve results significantly when working with such challenging samples.

How can researchers distinguish between signals from AT5G08690 and its close paralogs (AT5G08670 and AT5G08680) in experimental systems?

Distinguishing between highly similar ATP synthase beta subunit paralogs requires careful experimental design:

• Peptide Selection for Validation:

  • Identify unique peptide sequences that differ between the paralogs

  • Design parallel reaction monitoring (PRM) mass spectrometry assays targeting these unique peptides

  • Use synthetic peptide standards for absolute quantification

• Genetic Approach:

  • Utilize T-DNA insertion lines or CRISPR-Cas9 knockout lines for individual paralogs

  • Verify knockout efficiency using gene-specific primers

  • Compare antibody signal patterns between wild-type and mutant lines

• Epitope Analysis:

  • Determine if the antibody epitope is in a conserved or variable region

  • For epitopes in conserved regions, complementary approaches are necessary

  • Consider using RNA-level analysis (qRT-PCR with paralog-specific primers) to correlate with protein data

Table: Sequence Comparison of ATP Synthase Beta Subunit Paralogs

Since the AT5G08690 antibody cross-reacts with AT5G08670 and AT5G08680 due to high sequence similarity , researchers should be aware that signals represent the combined pool of all three paralogs unless complementary approaches are employed.

What considerations are important when using AT5G08690 antibody in ATP synthase knockdown studies?

When using AT5G08690 antibody in ATP synthase knockdown studies, several critical factors must be considered:

Research by Kerbler demonstrated that ATP synthase knockdown lines show differential responses to low temperature treatment, with varying effects on respiration rates depending on the degree of knockdown . AT5G08690 antibody provides a reliable tool for quantifying these changes at the protein level, enabling researchers to establish clear cause-effect relationships.

How should researchers interpret changes in ATP synthase subunit stoichiometry detected by AT5G08690 antibody under different experimental conditions?

Interpreting changes in ATP synthase subunit stoichiometry requires careful analytical consideration:

• Quantitative Analysis Approach:

  • Use a combination of western blotting (with AT5G08690 antibody) and selective reaction monitoring (SRM) mass spectrometry

  • Calculate absolute quantities of ATP synthase subunits using standard curves

  • Compare stoichiometric ratios between different subunits (e.g., beta:alpha, beta:gamma)

  • Normalize to established mitochondrial markers (e.g., porin, TOM40)

• Physiological Context Assessment:

  • Consider that altered stoichiometry may indicate:

    • Changes in ATP synthase assembly

    • Differential subunit turnover rates

    • Compensatory mechanisms for functional deficiencies

  • Correlate with ATP synthesis rates and respiratory coupling efficiency

• Statistical Validation:

  • Apply appropriate statistical tests (ANOVA with post-hoc tests)

  • Ensure sufficient biological replicates (minimum n=3, preferably n≥5)

  • Consider technical variability in antibody-based quantification

Research on plant responses to cold stress has revealed that while beta subunit abundance (detected by AT5G08690 antibody) may change moderately, the ratio between different ATP synthase subunits can shift significantly, suggesting complex regulatory mechanisms beyond simple upregulation or downregulation . These stoichiometric changes often correlate with altered ATP synthesis capacity and efficiency, providing insights into stress adaptation mechanisms.

What statistical approaches are most appropriate for analyzing western blot data generated using AT5G08690 antibody across multiple experimental conditions?

For robust statistical analysis of western blot data using AT5G08690 antibody:

• Normalization Strategies:

  • Use total protein normalization (stain-free gels or Ponceau S)

  • Alternatively, normalize to stable mitochondrial markers

  • Avoid using housekeeping proteins that may vary under experimental conditions

  • Consider multiple normalization approaches and compare results

• Statistical Tests Selection:

  • For comparing two conditions: Student's t-test or Mann-Whitney U test (for non-parametric data)

  • For multiple conditions: One-way ANOVA followed by Tukey's or Dunnett's post-hoc tests

  • For factorial designs: Two-way ANOVA to assess interaction effects

  • For time-course experiments: Repeated measures ANOVA or mixed-effects models

• Sample Size and Power Considerations:

  • Perform power analysis to determine appropriate sample size

  • Typically, n=4-6 biological replicates provides adequate statistical power

  • Include technical replicates to assess methodological variability

• Advanced Analytical Approaches:

  • Consider using principal component analysis (PCA) for multivariate data

  • Apply hierarchical clustering to identify patterns across multiple proteins and conditions

  • Use Volcano plots to visualize both magnitude and statistical significance of changes

These analytical approaches have been successfully applied in studies of mitochondrial protein dynamics during temperature stress, revealing statistically significant changes in ATP synthase abundance that correlate with physiological adaptations .

How can researchers integrate AT5G08690 antibody-based protein quantification with transcriptomic and metabolomic data to gain comprehensive insights into mitochondrial function?

Integrating AT5G08690 antibody-based protein data with other omics approaches enables comprehensive understanding of mitochondrial function:

• Multi-Omics Data Collection Strategy:

  • Perform parallel analyses on the same biological samples:

    • Protein quantification using AT5G08690 antibody (Western blot/SRM)

    • Transcript analysis (RNA-seq or qRT-PCR)

    • Metabolite profiling (LC-MS/GC-MS)

    • Lipid analysis for mitochondrial membrane components

• Correlation Analysis:

  • Calculate Pearson or Spearman correlation coefficients between:

    • Protein abundance (AT5G08690 signal)

    • mRNA levels of ATP synthase genes

    • ATP/ADP ratios and related metabolites

    • Membrane lipid composition

• Pathway Mapping:

  • Map all data onto known bioenergetic pathways

  • Identify regulatory nodes where transcriptional, translational, and post-translational regulation diverge

  • Use pathway enrichment analysis to identify coordinated responses

• Visualization and Integration Tools:

  • Apply dimension reduction techniques (PCA, t-SNE)

  • Create correlation networks to visualize relationships

  • Use heatmaps with hierarchical clustering to identify patterns

  • Consider machine learning approaches for pattern recognition

Table: Multi-Omics Integration Parameters for ATP Synthase Analysis

This integrated approach has revealed that changes in ATP synthase protein abundance (detected by AT5G08690 antibody) often precede alterations in membrane lipid composition during stress responses, highlighting the complex interplay between protein function and membrane environment in mitochondrial adaptation .