At5g47030 Antibody

What is At5g47030 and what role does it play in plant mitochondria?

At5g47030 is a gene locus in Arabidopsis thaliana that encodes a subunit of the ATP synthase complex, a critical component of the oxidative phosphorylation machinery in plant mitochondria. ATP synthase functions as the powerhouse enzyme that couples the electrochemical proton gradient (ΔμH+) across the inner mitochondrial membrane to the synthesis of ATP from ADP and inorganic phosphate (Pi). The protein plays an essential role in energy metabolism, particularly during stress responses such as cold acclimation. Research indicates that ATP synthase activity is significantly affected by temperature changes, potentially functioning as a respiratory bottleneck during chilling stress .

What are the key characteristics of antibodies targeting the At5g47030 protein?

Antibodies against the At5g47030 protein are typically polyclonal or monoclonal immunoglobulins designed to recognize specific epitopes of this ATP synthase subunit. High-quality antibodies exhibit several essential characteristics:

• High specificity for the target protein with minimal cross-reactivity

• Appropriate sensitivity for detection in various experimental conditions

• Consistent performance across different batches

• Validated functionality in multiple applications (Western blotting, immunoprecipitation, immunohistochemistry, etc.)

Researchers should verify these characteristics through validation experiments using positive and negative controls, including ATP synthase knockdown lines if available .

How can I verify the specificity of an At5g47030 antibody?

Verifying antibody specificity requires multiple complementary approaches:

• Western blot analysis comparing wild-type Arabidopsis samples with those of ATP synthase knockdown lines (such as dexamethasone-inducible RNAi lines targeting ATP synthase subunits)

• Peptide competition assays where the antibody is pre-incubated with the immunizing peptide

• Cross-reactivity testing against different plant species and tissues

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Use of multiple antibodies targeting different epitopes of the same protein

Significant differences in protein detection between wild-type and knockdown samples, particularly after treatments that alter ATP synthase expression (such as cold acclimation), provide strong evidence for antibody specificity .

What are the recommended protocols for using At5g47030 antibody in Western blot analysis?

For optimal Western blot analysis using the At5g47030 antibody:

• Sample preparation:

  • Extract mitochondria using established differential centrifugation methods

  • Resuspend isolated mitochondria in appropriate buffer (e.g., 0.3 M sucrose, 10 mM TES, 2 mM EDTA)

  • Determine protein concentration using Bradford or BCA assay

  • Denature samples with SDS loading buffer at 95°C for 5 minutes

• Gel electrophoresis and transfer:

  • Separate proteins on 12-15% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membranes at 100V for 1 hour

• Immunoblotting:

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

  • Incubate with primary At5g47030 antibody (1:1000-1:5000 dilution) overnight at 4°C

  • Wash 3× with TBS-T

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

  • Develop using ECL (enhanced chemiluminescence) detection

For quantitative analysis, consider using selected reaction monitoring (SRM) mass spectrometry as performed for ATP synthase subunits in the referenced research .

How can I use the At5g47030 antibody to study changes in ATP synthase abundance during stress responses?

To effectively study ATP synthase abundance changes during stress responses:

• Experimental design:

  • Subject plants to controlled stress conditions (e.g., cold acclimation at 4°C for 1-7 days)

  • Include time-course sampling points (e.g., 0, 1, 3, 5, 7 days)

  • Maintain appropriate controls (warm-grown plants)

• Sample processing:

  • Isolate mitochondria from each condition using density gradient centrifugation

  • Ensure equal protein loading across samples

• Quantitative analysis:

  • Perform Western blots with the At5g47030 antibody

  • Use digital imaging for densitometry analysis

  • Normalize to appropriate loading controls (e.g., porin, VDAC)

  • Consider complementary approaches such as SRM mass spectrometry

• Data interpretation:

  • Compare relative abundance across conditions

  • Correlate with functional measurements (respiratory rates, ATP synthesis)

This approach has been successfully used to demonstrate differential responses of ATP synthase subunits during cold acclimation and cold shock conditions .

What protocols are recommended for immunolocalization studies with the At5g47030 antibody?

For effective immunolocalization of At5g47030 in plant tissues:

• Tissue preparation:

  • Fix plant material in 4% paraformaldehyde in PBS

  • Embed in paraffin or prepare for cryo-sectioning

  • Cut 5-10 μm sections and mount on coated slides

• Immunostaining protocol:

  • Deparaffinize and rehydrate sections

  • Perform antigen retrieval (if necessary)

  • Block with 5% BSA in PBS for 1 hour

  • Incubate with At5g47030 antibody (1:100-1:500) overnight at 4°C

  • Wash 3× with PBS

  • Apply fluorescent secondary antibody (1:500) for 2 hours

  • Counterstain with DAPI for nuclear visualization

  • Mount with anti-fade medium

• Mitochondrial co-localization:

  • Include mitochondrial markers (e.g., COX antibodies)

  • Use appropriate fluorophore combinations for multi-channel imaging

• Controls:

  • Include secondary-only controls

  • Use tissues from ATP synthase knockdown lines as negative controls

  • Perform peptide competition controls

Confocal microscopy is recommended for optimal visualization of mitochondrial localization patterns.

What are common issues when using At5g47030 antibody and how can they be resolved?

For temperature-sensitive applications, note that ATP synthase activity varies significantly with temperature (Q10 values ~2.0), which may affect antibody binding efficiency in certain applications .

How should the At5g47030 antibody be stored and handled to maintain optimal activity?

For optimal antibody stability and performance:

• Storage conditions:

  • Store antibody aliquots at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles (limit to <5)

  • For working solutions, store at 4°C for up to 2 weeks

• Handling recommendations:

  • Centrifuge vials briefly before opening

  • Prepare working dilutions immediately before use

  • Add preservatives (0.02% sodium azide) for solutions stored at 4°C

  • Keep on ice during experimental procedures

• Quality control:

  • Document lot numbers and expiration dates

  • Periodically test against reference samples

  • Store small aliquots of positive control samples at -80°C

Following these practices will help maintain antibody specificity and sensitivity, ensuring reproducible results across experiments.

How can the At5g47030 antibody be used to investigate ATP synthase coupling efficiency during environmental stress?

Investigating ATP synthase coupling efficiency during stress requires combining antibody-based quantification with functional assays:

• Experimental approach:

  • Isolate intact mitochondria from plants under various stress conditions

  • Maintain sample integrity to prevent uncoupling

  • Use parallel samples for protein quantification and functional assays

• Functional measurements:

  • Measure ATP/O ratios at different temperatures

  • Determine membrane potential (ΔΨm) using fluorescent dyes (e.g., TMRM)

  • Assess respiration rates in different states (State 3, State 4)

  • Calculate respiratory control ratios (RCRs)

• Protein analysis:

  • Quantify At5g47030 and other ATP synthase subunits using the antibody

  • Correlate subunit abundance with functional parameters

  • Analyze post-translational modifications that may affect coupling

• Data integration:

  • Plot respiratory capacity against ATP synthase abundance

  • Determine temperature coefficients (Q10) for enzyme activities

  • Create mathematical models of ATP synthase efficiency under stress

This integrated approach can reveal how changes in ATP synthase composition affect its function as a potential respiratory bottleneck during environmental stress .

What approaches can be used to study interactions between At5g47030 and other mitochondrial proteins?

Several sophisticated approaches can be employed to study protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use At5g47030 antibody for pull-down experiments

  • Analyze co-precipitated proteins by mass spectrometry

  • Verify interactions with reciprocal Co-IP experiments

• Proximity labeling approaches:

  • Express BioID or APEX2 fusions of At5g47030

  • Identify proximal proteins through biotinylation

  • Confirm interactions using the At5g47030 antibody

• Blue Native PAGE:

  • Separate native protein complexes

  • Perform second-dimension SDS-PAGE

  • Detect At5g47030 and interacting partners with specific antibodies

• Fluorescence microscopy techniques:

  • Implement FRET or BiFC to visualize interactions in vivo

  • Use the At5g47030 antibody to validate expression

  • Perform co-localization studies with other mitochondrial proteins

• Cross-linking mass spectrometry:

  • Apply protein cross-linkers to stabilize transient interactions

  • Enrich for At5g47030-containing complexes using the antibody

  • Identify cross-linked peptides by specialized mass spectrometry

These methods can reveal how At5g47030 interacts with other ATP synthase subunits and potentially with regulatory proteins during stress responses .

How can At5g47030 antibody be used to investigate changes in mitochondrial membrane composition during temperature stress?

To investigate mitochondrial membrane composition changes during temperature stress:

• Isolation of mitochondria and membrane fractions:

  • Extract mitochondria from plants subjected to different temperature treatments

  • Separate inner and outer membranes using established fractionation techniques

  • Verify fraction purity using marker proteins

• Protein-lipid association analysis:

  • Perform lipidomic analysis of mitochondrial membranes

  • Use the At5g47030 antibody to localize the protein within membrane fractions

  • Employ detergent resistance assays to identify lipid raft associations

• Correlation of protein distribution with membrane properties:

  • Measure membrane fluidity at different temperatures

  • Quantify cardiolipin and other mitochondria-specific lipids

  • Correlate At5g47030 distribution with changes in specific lipid classes

• Functional implications:

  • Assess ATP synthase activity in relation to membrane composition

  • Measure proton leak across membranes with different compositions

  • Determine how lipid environment affects protein stability and function

Research has shown significant changes in mitochondrial lipid composition during cold acclimation, which may directly affect ATP synthase function and stability .

What are the best approaches for studying At5g47030 post-translational modifications using antibody-based methods?

For comprehensive analysis of post-translational modifications (PTMs):

• PTM-specific antibody approaches:

  • Use general PTM antibodies (phospho-, acetyl-, etc.) after At5g47030 immunoprecipitation

  • Develop modification-specific antibodies for known PTM sites

  • Perform Western blots under conditions that preserve labile modifications

• Mass spectrometry-based strategies:

  • Immunoprecipitate At5g47030 using the specific antibody

  • Digest with appropriate proteases (trypsin, chymotrypsin, etc.)

  • Analyze PTMs using high-resolution mass spectrometry

  • Quantify modification stoichiometry with targeted methods

• Functional correlation:

  • Compare PTM profiles between different stress conditions

  • Correlate modifications with ATP synthase activity measurements

  • Generate site-directed mutants to assess PTM significance

• Temporal dynamics:

  • Track modification changes during stress imposition and recovery

  • Correlate with known signaling pathways activated during stress

PTMs may play crucial roles in regulating ATP synthase activity during temperature shifts, particularly affecting its sensitivity to inhibitors and coupling efficiency .

How can genetic variation in At5g47030 across plant ecotypes be studied using antibody-based methods?

To investigate genetic variation in At5g47030 across ecotypes:

• Sample collection and preparation:

  • Obtain diverse Arabidopsis ecotypes (e.g., Col-0, T1110, etc.)

  • Grow under identical conditions to minimize environmental effects

  • Isolate mitochondria using consistent protocols

• Antibody-based analysis:

  • Perform Western blots to compare protein abundance across ecotypes

  • Assess epitope conservation through signal intensity comparison

  • Combine with mass spectrometry for deeper sequence analysis

• Functional characterization:

  • Measure ATP synthase activity in different ecotypes

  • Determine temperature response curves (Q10 values)

  • Assess respiratory parameters (State 3, State 4, RCR)

• Data integration:

  • Correlate protein variations with functional differences

  • Link to known SNPs in the At5g47030 gene

  • Develop predictive models for structure-function relationships

This approach has been successful in identifying ecotypes with differential responses to temperature stress, such as the T1110 ecotype which shows higher uncoupled respiration rates at 4°C compared to Col-0 .

What statistical approaches are most appropriate for analyzing At5g47030 antibody-based quantification data?

Proper statistical analysis of antibody-based quantification requires:

• Experimental design considerations:

  • Include sufficient biological replicates (minimum n=3)

  • Plan for appropriate technical replicates

  • Incorporate randomization and blinding where possible

• Data normalization approaches:

  • Normalize to loading controls (VDAC, porin)

  • Consider total protein normalization (Ponceau, SYPRO Ruby)

  • Apply appropriate transformations for non-normal data

• Statistical tests for different scenarios:

  • For comparing two conditions: t-test or non-parametric equivalent

  • For multiple conditions: ANOVA with appropriate post-hoc tests

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

• Advanced multivariate methods:

  • Principal Component Analysis (PCA) for pattern recognition

  • Hierarchical clustering for sample relationship visualization

  • Partial Least Squares Discriminant Analysis (PLS-DA) for group separation

• Visualization best practices:

  • Display individual data points alongside means

  • Use error bars representing standard error of the mean (SEM)

  • Create heatmaps for visualizing complex relationships

Implementing these robust statistical approaches will enhance the reliability and reproducibility of At5g47030 antibody-based research findings .

How can At5g47030 antibody be used in combination with respiratory measurements to create predictive models of plant stress responses?

Integrating antibody-based quantification with respiratory measurements enables development of predictive models:

• Data collection strategy:

  • Measure multiple parameters on parallel samples:

    • At5g47030 abundance (antibody-based)

    • Respiratory rates (O2 consumption)

    • Membrane potential (fluorescent probes)

    • ATP production rates

    • ROS production

  • Include time-course measurements during stress imposition and recovery

• Data integration approaches:

  • Calculate correlation matrices between all parameters

  • Develop mathematical models of respiratory control

  • Apply machine learning algorithms to identify predictive patterns

• Model validation:

  • Test predictions on new stress conditions or genetic variants

  • Perform sensitivity analysis to identify critical parameters

  • Validate key relationships with genetic manipulation (e.g., ATP synthase knockdown lines)

• Practical applications:

  • Predict responses to novel stress combinations

  • Identify potential intervention points for improving stress tolerance

  • Guide breeding efforts for stress-resistant varieties

How might the At5g47030 antibody be utilized in single-cell or subcellular studies of mitochondrial heterogeneity?

Emerging technologies enable unprecedented resolution in studying mitochondrial heterogeneity:

• Single-cell proteomics approaches:

  • Use antibody-based flow cytometry of isolated mitochondria

  • Implement microfluidic sorting of mitochondrial populations

  • Apply single-cell Western blotting techniques

  • Develop CITE-seq approaches for mitochondrial proteins

• Super-resolution microscopy applications:

  • Employ STORM or PALM imaging with labeled At5g47030 antibodies

  • Perform correlative light and electron microscopy (CLEM)

  • Implement expansion microscopy for improved spatial resolution

  • Track dynamic changes in ATP synthase distribution during stress

• Mitochondrial sub-compartment analysis:

  • Study ATP synthase localization in cristae vs. boundary membranes

  • Investigate protein-lipid domains within mitochondrial membranes

  • Examine co-localization with other respiratory complexes

• Heterogeneity assessment:

  • Quantify variation in At5g47030 abundance between individual mitochondria

  • Correlate with functional parameters (membrane potential, ROS production)

  • Track changes in heterogeneity during stress responses

These approaches can reveal previously unappreciated diversity in mitochondrial populations and their differential responses to environmental challenges .

What considerations are important when developing new antibodies or antibody-based tools for studying At5g47030 and related proteins?

Development of next-generation antibody tools requires careful consideration:

• Epitope selection strategies:

  • Target conserved regions for cross-species applications

  • Identify accessible epitopes based on structural information

  • Consider generating antibodies against post-translationally modified sites

  • Design epitopes that distinguish between highly similar ATP synthase isoforms

• New antibody formats:

  • Develop single-domain antibodies (nanobodies) for improved penetration

  • Create recombinant antibody fragments with enhanced stability

  • Engineer bifunctional antibodies for proximity labeling applications

  • Produce conformation-specific antibodies to detect functional states

• Validation requirements:

  • Implement multiplexed validation using orthogonal methods

  • Test in multiple species if cross-reactivity is desired

  • Validate in knockout/knockdown models

  • Assess specificity across diverse experimental conditions

• Application optimization:

  • Develop antibodies optimized for specific applications (ChIP, IP-MS, etc.)

  • Create tagged antibodies for direct detection without secondary antibodies

  • Develop antibody pairs for proximity ligation assays

These considerations will guide the development of more specific, sensitive, and versatile tools for studying ATP synthase biology in plants .