At3g08610 Antibody

What types of AT3G08610 antibodies are available for plant research?

Several types of antibodies targeting AT3G08610 are available for research applications:

These antibodies undergo application-specific validation to ensure their performance in the indicated experimental contexts. Each antibody targets different epitopes of the AT3G08610 protein, allowing researchers to select the most appropriate reagent for their specific experimental needs .

How should AT3G08610 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of AT3G08610 antibodies are crucial for maintaining their activity:

• Storage temperature: Upon receipt, store at -20°C or -80°C

• Buffer composition: Most are supplied in a buffer containing:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 as preservative

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles by aliquoting antibodies upon receipt

• Working dilutions: Prepare fresh working dilutions immediately before use

• Shipping conditions: Antibodies are typically shipped at 4°C but should be transferred to recommended storage temperature immediately upon receipt

Following these guidelines ensures the maintenance of antibody binding efficiency and specificity throughout your experimental timeline, preventing degradation that could compromise experimental results .

What are the validated applications for AT3G08610 antibodies?

AT3G08610 antibodies have been validated for specific applications through rigorous testing:

The antibodies are purified using antigen affinity methods to ensure specificity for the target protein . It's important to note that while these are the primary validated applications, researchers have also adapted these antibodies for other techniques including:

• Immunohistochemistry (IHC) in plant tissues

• Immunoprecipitation (IP) to study protein interactions

• Blue Native PAGE to study Complex I assembly

Each application requires specific optimization steps for maximum sensitivity and specificity .

How can AT3G08610 antibodies be used to study mitochondrial Complex I assembly in plants?

AT3G08610 antibodies provide powerful tools for investigating Complex I assembly mechanisms:

• Blue Native PAGE combined with Western blot analysis:

  • Solubilize mitochondrial membranes with digitonin (1%)

  • Separate native complexes on 3-12% gradient gels

  • Transfer to PVDF membranes

  • Probe with AT3G08610 antibodies to identify assembly intermediates

  • Compare patterns between wild-type and mutant plants

• Co-immunoprecipitation to identify interaction partners:

  • Crosslink protein complexes in isolated mitochondria

  • Solubilize with appropriate detergents

  • Immunoprecipitate with AT3G08610 antibodies

  • Identify interacting proteins by mass spectrometry

• Pulse-chase experiments to study assembly kinetics:

  • Label newly synthesized proteins with isotopic amino acids

  • Track incorporation of AT3G08610 into Complex I over time using immunoprecipitation

  • Analyze by autoradiography or mass spectrometry

• Immunogold electron microscopy:

  • Localize AT3G08610 within mitochondrial subcompartments

  • Quantify gold particle distribution in different physiological conditions

These approaches have revealed that plant Complex I assembly follows distinct pathways from those in animal systems, with unique plant-specific subunits like AT3G08610 playing critical roles in the process.

What cross-reactivity profiles do AT3G08610 antibodies exhibit across different plant species?

AT3G08610 antibodies show varying cross-reactivity profiles that should be considered when designing experiments with non-Arabidopsis species:

When working with species not listed, sequence homology analysis should be performed:

• Align the AT3G08610 sequence from Arabidopsis with the orthologous sequence from your species of interest

• Assess conservation of epitope regions (especially for monoclonal antibodies)

• Validate cross-reactivity experimentally with appropriate controls

Research has demonstrated successful application of these antibodies in comparative studies of respiratory complexes across plant species, revealing evolutionary conservation of Complex I structure despite some species-specific variations .

What controls should be included when using AT3G08610 antibodies in experimental protocols?

Rigorous experimental design for AT3G08610 antibody applications requires the following controls:

• Positive controls:

  • Isolated mitochondria from wild-type Arabidopsis

  • Recombinant AT3G08610 protein (if available)

• Negative controls:

  • Preimmune serum (for polyclonal antibodies)

  • Isotype control antibodies (for monoclonals)

  • AT3G08610 knockout/knockdown mutant samples

  • Secondary antibody-only controls

• Specificity controls:

  • Peptide competition assays using immunizing peptides

  • Multiple antibodies targeting different epitopes

  • Comparative signal analysis across tissue types

• Technical controls:

  • Loading controls (alternative mitochondrial proteins)

  • Molecular weight markers

  • Degradation controls (freshly isolated vs. stored samples)

Implementation of these controls enables accurate interpretation of experimental results and validation of antibody specificity, which is particularly important when studying proteins like AT3G08610 that exist within large complexes with structural similarities to other mitochondrial components.

How do expression levels of AT3G08610 vary across plant development and stress conditions?

Understanding AT3G08610 expression patterns provides context for antibody-based experiments:

Expression is significantly modulated by:

• Oxidative stress (2-3 fold increase)

• Cold stress (1.5-2 fold increase)

• Nutrient limitation (nitrogen: decreased; phosphorus: variable)

AT3G08610 antibodies have been instrumental in revealing these expression patterns, which correlate with increased respiratory demands during seed development and stress responses. The antibodies can detect both the basal and induced levels of the protein, making them valuable tools for studying mitochondrial adaptation to environmental conditions .

What are the optimized protocols for using AT3G08610 antibodies in Western blotting?

For optimal Western blot results with AT3G08610 antibodies:

• Sample preparation:

  • Isolate mitochondria using differential centrifugation

  • Resuspend in buffer containing 0.3M sucrose, 10mM TES, 10mM MOPS, pH 7.2

  • Add protease inhibitor cocktail

  • Solubilize with 1% digitonin or 1% n-dodecyl-β-D-maltoside

• Gel electrophoresis:

  • Use Tricine-SDS-PAGE (16%) for better resolution of small proteins

  • Load 10-20 μg mitochondrial protein per lane

  • Include molecular weight markers covering 5-15 kDa range

• Transfer conditions:

  • Transfer to PVDF membranes (0.2 μm pore size)

  • Use semi-dry transfer: 1.5 mA/cm² for 45 minutes

  • Transfer buffer: 25mM Tris, 192mM glycine, 20% methanol, 0.02% SDS

• Antibody incubation:

  • Block with 5% non-fat milk in TBS-T for 1 hour at room temperature

  • Primary antibody dilution: 1:2000 in 2.5% milk/TBS-T

  • Incubate overnight at 4°C

  • Wash 3×15 minutes with TBS-T

  • Secondary antibody: 1:5000 HRP-conjugated anti-rabbit/mouse IgG

• Detection:

  • Use enhanced chemiluminescence (ECL)

  • Expected band size: approximately 7-10 kDa

  • Exposure time: 1-5 minutes for standard ECL systems

This protocol has been optimized based on research addressing the challenges of detecting small mitochondrial proteins like AT3G08610, which can otherwise be difficult to resolve and visualize.

How can AT3G08610 antibodies be used to investigate the unfolded protein response in plant mitochondria?

AT3G08610 antibodies provide valuable tools for studying mitochondrial stress responses:

• Monitoring protein accumulation during UPR:

  • Treat plants with electron transport inhibitors (antimycin A, rotenone)

  • Isolate mitochondria at different time points

  • Quantify AT3G08610 levels by Western blot

  • Compare with expression of known UPR marker genes

• Subcellular localization during stress:

  • Perform immunofluorescence microscopy with AT3G08610 antibodies

  • Co-stain with markers for different mitochondrial subcompartments

  • Analyze changes in localization pattern during stress conditions

• Interaction studies during UPR:

  • Perform co-immunoprecipitation with AT3G08610 antibodies

  • Compare interacting partners under normal vs. stress conditions

  • Identify stress-specific interactions

Research using this approach has shown that Complex I subunits like AT3G08610 exhibit altered abundance and interaction patterns during mitochondrial stress, suggesting regulatory roles beyond their structural functions in electron transport .

What is the role of recombinant antibody technology in improving AT3G08610 antibody specificity?

Recombinant antibody technology has significantly improved AT3G08610 antibody development:

• Enhancement approaches:

  • Generation of single-chain variable fragments (scFv)

  • Production of camelid-derived single-domain antibodies (nanobodies)

  • Creation of bispecific antibodies targeting multiple epitopes

• Advantages for plant mitochondrial research:

  • Increased specificity for closely related Complex I subunits

  • Better penetration into densely packed mitochondrial membranes

  • Reduced background in immunolocalization studies

  • Enhanced stability under various experimental conditions

• Implementation methods:

  • Phage display selection against purified AT3G08610 protein

  • Yeast surface display for affinity maturation

  • Bacterial or plant-based expression systems for antibody production

Studies have demonstrated that nanobodies derived from camelid immunization show particularly strong potential for recognizing AT3G08610 in its native conformation within Complex I, as their small size allows access to epitopes that might be sterically hindered from conventional antibodies .

How can anti-drug antibody (ADA) analysis methods be adapted to validate AT3G08610 antibody specificity?

Techniques from therapeutic antibody development can be adapted for validating research antibodies:

• Electrochemiluminescence-based binding assays:

  • Immobilize recombinant AT3G08610 on plates

  • Test antibody binding with ECL detection

  • Quantify binding affinity and specificity

  • Compare different antibody lots for consistency

• Epitope analysis:

  • Generate truncated fragments of AT3G08610

  • Determine binding to different regions

  • Map precise epitopes using peptide arrays

  • Verify accessibility of epitopes in native protein

• Neutralization assays:

  • Test antibody inhibition of AT3G08610 function in vitro

  • Use functional assays like NADH oxidation activity

  • Quantify degree of inhibition at different antibody concentrations

This methodological crossover from therapeutic antibody validation to research antibody characterization provides rigorous quality control measures that ensure reproducible results in plant mitochondrial research .