At4g20930 Antibody

How do At4g20930 antibodies differ from other plant protein antibodies?

At4g20930 antibodies are specifically designed to target the AtHDH1 protein in Arabidopsis thaliana. Unlike antibodies for structural proteins or signaling molecules that may be conserved across multiple plant species, AtHDH1 antibodies require careful validation due to the potential sequence variations in metabolic enzymes across plant species. These antibodies typically need to recognize specific epitopes on the 3-hydroxyisobutyrate dehydrogenase protein, which may be located at the N-terminus, C-terminus, or internal regions. The effectiveness of these antibodies can be particularly dependent on protein conformation, especially since AtHDH1 functions in mitochondria, which may affect epitope accessibility in different experimental contexts .

What are the key applications for At4g20930 antibodies in plant science?

At4g20930 antibodies serve several critical functions in plant science research:

• Localization studies: Determining the subcellular localization of AtHDH1 in plant cells, particularly its association with mitochondria where BCAA catabolism occurs.

• Protein expression analysis: Monitoring AtHDH1 protein levels during different developmental stages or stress conditions, particularly during carbohydrate limitation.

• Protein-protein interaction studies: Investigating how AtHDH1 interacts with other components of the BCAA catabolic pathway.

• Functional validation: Confirming the presence or absence of the protein in knockout or knockdown mutants.

• Post-translational modification analysis: Examining how AtHDH1 might be regulated through modifications like phosphorylation under different metabolic conditions .

How should researchers select the appropriate At4g20930 antibody for their specific experiment?

When selecting an At4g20930 antibody, researchers should consider:

• Experimental technique: Different antibodies may be optimized for Western blot, immunoprecipitation, or immunofluorescence. For example, some epitopes may be accessible only in denatured conditions (Western blot) but not in native conditions (immunoprecipitation).

• Target region: Consider whether N-terminal, C-terminal, or middle region antibodies are most appropriate:

  • N-terminal antibodies may be useful if the protein undergoes C-terminal processing

  • C-terminal antibodies may be preferred if N-terminal modifications occur

  • Middle region antibodies might offer better accessibility in folded proteins

• Cross-reactivity: Verify whether the antibody cross-reacts with homologous proteins in your experimental system, especially if studying multiple plant species.

• Validation data: Review existing validation data, particularly ELISA titers and detection limits on Western blots. Effective At4g20930 antibodies typically show ELISA titers around 10,000, corresponding to approximately 1 ng detection sensitivity on Western blots .

What validation steps are essential before using an At4g20930 antibody in research?

Before proceeding with experiments, comprehensive validation of At4g20930 antibodies should include:

• Western blot with positive controls: Use purified recombinant AtHDH1 protein or extracts from wild-type Arabidopsis thaliana plants known to express the protein.

• Negative control testing: Test the antibody against extracts from confirmed At4g20930 knockout plants or tissues where the protein is not expressed.

• Peptide competition assay: Pre-incubate the antibody with excess synthetic peptide representing the epitope to confirm specificity.

• Cross-reactivity assessment: Test against related proteins or extracts from other plant species if your research involves comparative analysis.

• Reproducibility verification: Ensure consistent results across multiple protein preparations and antibody lots.

• Background signal evaluation: Assess non-specific binding by comparing secondary-only controls against full antibody application.

This rigorous validation is particularly important for metabolic enzymes like AtHDH1, which may share structural similarities with other dehydrogenases .

What are the differences between monoclonal and polyclonal antibodies for At4g20930 detection?

For At4g20930 research, monoclonal antibodies provide advantages in experimental reproducibility and specificity. Combinations of individual monoclonal antibodies against different epitopes (as shown in search result for other proteins) can offer increased sensitivity while maintaining specificity. For example, a combination approach might use multiple monoclonal antibodies targeting different regions of AtHDH1 to enhance detection capabilities while maintaining the low background benefits of monoclonals .

How can At4g20930 antibodies be optimized for Western blot applications?

Optimizing At4g20930 antibodies for Western blot requires careful attention to several parameters:

• Protein extraction: Use extraction buffers containing appropriate protease inhibitors to prevent degradation of AtHDH1. Since this protein functions in mitochondria, consider mitochondrial enrichment protocols to increase detection sensitivity.

• Denaturation conditions: Test both reducing and non-reducing conditions, as disulfide bonds might affect epitope accessibility in AtHDH1.

• Transfer optimization:

  • Use PVDF membranes for higher protein binding capacity

  • For AtHDH1 (approximately 35-40 kDa), standard transfer conditions (100V for 1 hour) are typically sufficient

  • Consider wet transfer for more quantitative analysis

• Blocking optimization:

  • Test both BSA and non-fat milk as blocking agents (5% concentration)

  • BSA often works better for phospho-specific antibodies if studying AtHDH1 post-translational modifications

• Antibody dilution optimization:

  • Start with manufacturer's recommended concentration

  • Typically 1:1000 to 1:5000 for primary antibody

  • Incubate overnight at 4°C for optimal sensitivity

• Signal detection:

  • For low abundance proteins, consider enhanced chemiluminescence (ECL) with longer exposure times

  • Fluorescent secondary antibodies allow for more precise quantification

• Positive controls: Include recombinant AtHDH1 or extracts from tissues known to express high levels of the protein .

What are the best practices for immunolocalization of At4g20930/AtHDH1 in plant tissues?

For effective immunolocalization of AtHDH1 in plant tissues:

• Fixation optimization:

  • Test both formaldehyde (4%) and paraformaldehyde (4%) fixation

  • Optimize fixation time (typically 2-4 hours) to balance tissue preservation and epitope accessibility

  • Consider dual fixation with glutaraldehyde (0.1-0.5%) for better ultrastructural preservation when examining mitochondrial localization

• Tissue preparation:

  • For fresh tissue sections: Use vibratome sectioning (50-100 μm)

  • For fixed tissues: Consider paraffin embedding with sections of 5-10 μm

  • For subcellular resolution: Consider cryosectioning (10-20 μm)

• Antigen retrieval:

  • Use citrate buffer (pH 6.0) heated to 95°C for 10-20 minutes

  • Cool slowly to room temperature before antibody application

• Antibody optimization:

  • Dilution series (typically 1:50 to 1:500) to determine optimal concentration

  • Extended incubation (overnight at 4°C) often improves signal-to-noise ratio

  • Use antibody combinations targeting different regions of AtHDH1 for confirmation

• Controls:

  • Negative control: Secondary antibody only

  • Competition control: Pre-absorb primary antibody with excess antigen

  • Biological control: At4g20930 knockout plant tissues

• Counterstaining:

  • DAPI for nuclear visualization

  • MitoTracker for mitochondrial co-localization studies (particularly important for AtHDH1)

• Confocal microscopy settings:

  • Z-stack acquisition to capture the full cellular distribution

  • Appropriate laser power to minimize photobleaching

  • Consistent settings between experimental and control samples .

How can researchers quantify At4g20930/AtHDH1 protein levels accurately in plant samples?

Accurate quantification of AtHDH1 protein requires rigorous methodology:

• Sample preparation standardization:

  • Harvest tissues at consistent developmental stages and times of day

  • Flash-freeze in liquid nitrogen immediately after collection

  • Process all experimental samples simultaneously

• Protein extraction optimization:

  • Use extraction buffers containing proper detergents (e.g., 1% Triton X-100)

  • Include protease inhibitor cocktail to prevent degradation

  • Consider mitochondrial isolation procedures for enrichment

• Quantification methods:

  • Western blot with densitometry:

    • Include purified recombinant AtHDH1 standards (5-50 ng) for calibration curve

    • Use housekeeping proteins (actin, tubulin) as loading controls

    • Image using a calibrated imaging system with linear dynamic range

  • ELISA-based quantification:

    • Develop sandwich ELISA using two antibodies recognizing different epitopes

    • Generate standard curve using purified recombinant protein

    • Process all samples in triplicate

  • Mass spectrometry-based approaches:

    • Selected reaction monitoring (SRM) for absolute quantification

    • Use isotope-labeled peptide standards for AtHDH1-specific peptides

    • Perform protein digestion and extraction with high consistency

• Data analysis:

  • Normalize to total protein content (determined by Bradford or BCA assay)

  • For comparative studies, express results as fold-change relative to control

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

• Validation of quantification:

  • Compare results using multiple antibodies targeting different regions

  • Confirm trends using mRNA quantification (qRT-PCR)

  • Verify specificity using knockout/knockdown lines as negative controls .

What are common issues when working with At4g20930 antibodies, and how can they be resolved?

Additionally, for AtHDH1 specifically, consider:

• Testing both membrane fractions and soluble fractions, as the protein's association with mitochondrial membranes may vary

• Using dual extraction methods (native and denaturing) if protein conformation affects antibody binding

• Employing recombinant AtHDH1 as a positive control to verify antibody functionality .

How can researchers study At4g20930/AtHDH1 protein-protein interactions?

To investigate AtHDH1 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use At4g20930 antibodies conjugated to magnetic or agarose beads

  • Extract proteins under non-denaturing conditions to preserve interactions

  • Verify specificity with IgG control and At4g20930 knockout samples

  • Identify interacting partners through mass spectrometry

  • Confirm key interactions with reverse Co-IP using antibodies against suspected partners

• Proximity labeling approaches:

  • Generate BioID or TurboID fusions with AtHDH1

  • Express in Arabidopsis through stable transformation

  • Induce proximity-dependent biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate with Co-IP or other interaction methods

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs of AtHDH1 with split YFP/GFP fragments

  • Co-express with candidate interactors fused to complementary fragments

  • Visualize reconstituted fluorescence in planta using confocal microscopy

  • Include appropriate controls (unfused fragments, known non-interactors)

• Förster Resonance Energy Transfer (FRET):

  • Generate donor-acceptor fluorophore pairs (e.g., CFP-AtHDH1 and YFP-candidate)

  • Express in plant cells and measure energy transfer using spectral imaging

  • Calculate FRET efficiency to estimate interaction strength

  • Perform acceptor photobleaching to confirm genuine FRET signals

• Yeast two-hybrid (Y2H) assays:

  • Create bait constructs with AtHDH1 and screen against cDNA libraries

  • Verify positive interactions with targeted Y2H assays

  • Confirm with in planta methods to rule out false positives

• Analytical size exclusion chromatography:

  • Analyze native protein complexes from plant extracts

  • Identify fractions containing AtHDH1 using Western blotting

  • Analyze co-eluting proteins by mass spectrometry

  • Compare profiles between wild-type and stress conditions .

What are the considerations for studying AtHDH1 under different metabolic stress conditions?

When investigating AtHDH1 under metabolic stress conditions:

How can At4g20930 antibodies contribute to understanding the evolutionary conservation of BCAA metabolism across plant species?

At4g20930 antibodies can provide valuable insights into the evolutionary conservation of branched-chain amino acid metabolism through:

• Cross-species immunoblotting:

  • Test antibody recognition across diverse plant lineages (monocots, dicots, gymnosperms, bryophytes)

  • Compare apparent molecular weights to identify potential structural modifications

  • Quantify relative protein abundance to identify species with enhanced BCAA metabolism

• Comparative immunolocalization:

  • Examine subcellular localization patterns across species

  • Identify potential differences in tissue-specific expression

  • Correlate with habitat-specific metabolic adaptations

• Epitope conservation analysis:

  • Map regions of high and low recognition by specific antibodies

  • Identify conserved functional domains across species

  • Use information to design broadly cross-reactive antibodies for comparative studies

• Structure-function relationship studies:

  • Combine antibody epitope mapping with protein modeling

  • Identify conserved regions that might be essential for catalytic activity

  • Correlate structural conservation with substrate specificity

• Stress response comparison:

  • Examine AtHDH1 protein levels under identical stress conditions across species

  • Correlate with ecological adaptations and stress tolerance

  • Identify species-specific regulatory mechanisms

• Co-evolution with interacting proteins:

  • Use antibodies to isolate protein complexes from different species

  • Compare interacting partners to identify conserved and species-specific interactions

  • Reconstruct evolutionary history of BCAA catabolic complexes .

What approaches can be used to study post-translational modifications of AtHDH1?

To investigate post-translational modifications (PTMs) of AtHDH1:

• Modification-specific antibodies:

  • Generate or acquire antibodies specific to phosphorylated, acetylated, or ubiquitinated AtHDH1

  • Perform Western blots to detect changes in modification status under different conditions

  • Use competition assays with modified and unmodified peptides to confirm specificity

• Mass spectrometry-based PTM mapping:

  • Immunoprecipitate AtHDH1 using validated antibodies

  • Perform tryptic digestion followed by LC-MS/MS analysis

  • Use neutral loss scanning for phosphorylation

  • Employ electron transfer dissociation (ETD) for modification site identification

  • Compare PTM patterns between different stress conditions

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Use AtHDH1 antibodies to identify different protein species

  • Compare spot patterns after phosphatase treatment

• Mobility shift assays:

  • Use Phos-tag acrylamide gels to separate phosphorylated from non-phosphorylated forms

  • Confirm phosphorylation with lambda phosphatase treatment

  • Detect with standard AtHDH1 antibodies

• Site-directed mutagenesis validation:

  • Identify potential modification sites through in silico analysis and MS data

  • Generate site-specific mutants (S/T→A for phosphorylation, K→R for acetylation/ubiquitination)

  • Express in At4g20930 knockout background

  • Assess functional consequences on enzyme activity and stress responses

• PTM-specific functional assays:

  • Compare enzyme kinetics before and after treatment with modifying/demodifying enzymes

  • Assess subcellular localization changes dependent on modification status

  • Determine if modifications affect protein-protein interactions .

How can researchers integrate At4g20930/AtHDH1 antibody data with multi-omics approaches for systems biology analysis?

For comprehensive systems biology analysis integrating AtHDH1 antibody data:

• Multi-level data acquisition:

  • Proteomics: AtHDH1 abundance and PTMs using antibody-based enrichment

  • Transcriptomics: RNA-seq to profile gene expression networks

  • Metabolomics: Targeted analysis of BCAA-related metabolites

  • Interactomics: Identify protein interaction networks using antibody-based pull-downs

  • Phenomics: Quantitative phenotyping of growth responses

• Temporal and spatial resolution:

  • Tissue-specific sampling using microdissection followed by antibody-based detection

  • Time-course experiments with consistent sampling intervals

  • Subcellular fractionation to track protein movement between compartments

• Data integration frameworks:

  • Correlation networks between protein abundance and metabolite levels

  • Causal modeling to infer regulatory relationships

  • Flux balance analysis incorporating enzyme abundance data

  • Pathway enrichment analysis incorporating multi-omics data

• Validation experiments:

  • Use genetic perturbations (knockdown, overexpression) to test model predictions

  • Apply specific metabolic inhibitors to verify pathway dependencies

  • Perform in vitro enzyme assays to confirm predicted activities

• Visualization and interpretation tools:

  • Pathway mapping with integrated multi-omics data

  • Temporal visualization of pathway dynamics

  • Network analysis to identify regulatory hubs

• Cross-species comparative analysis:

  • Map findings to orthologous systems in crop species

  • Identify conserved and divergent regulatory mechanisms

  • Translate fundamental insights into applied agricultural contexts

• Machine learning approaches:

  • Train predictive models using integrated datasets

  • Identify key variables predicting stress responses

  • Generate testable hypotheses for experimental validation .