GA2OX7 Antibody

What is GA2OX7 and why is it significant in plant research?

GA2OX7 belongs to the class III gibberellin 2-oxidases that catalyze the conversion of C20-GAs (such as GA12) to form GA110, effectively reducing bioactive gibberellin levels in plants. This enzyme plays crucial roles in various physiological processes including hypocotyl elongation, flowering time regulation, and stress responses. GA2OX7 is particularly significant as it acts in the restriction of root elongation under salt stress and in touch-induced growth repression of shoots . The enzyme contains distinctive DIOX_N and 2OG-FeII_Oxy domains and localizes to both the nucleus and cytoplasm, making it an important target for antibody-based detection in plant developmental studies .

What characteristics should researchers consider when selecting GA2OX7 antibodies?

When selecting antibodies against GA2OX7, researchers should consider:

• Epitope specificity - Target regions unique to GA2OX7 to avoid cross-reactivity with other GA2OX family members

• Species cross-reactivity - Determine if the antibody recognizes GA2OX7 from your plant species of interest (e.g., Arabidopsis, grape)

• Application compatibility - Verify suitability for intended applications (Western blot, immunohistochemistry, ChIP)

• Domain recognition - Consider antibodies targeting conserved functional domains (DIOX_N and 2OG-FeII_Oxy)

• Subcellular localization detection - Ensure the antibody can detect both nuclear and cytoplasmic pools of GA2OX7

Validation using recombinant GA2OX7 protein or extracts from plants with known GA2OX7 expression levels is essential before proceeding with experiments.

How should sample preparation be optimized for GA2OX7 detection?

Optimal sample preparation for GA2OX7 antibody applications requires:

• Rapid tissue harvesting and flash-freezing in liquid nitrogen to preserve protein integrity

• Homogenization in buffer containing appropriate protease inhibitors (GA2OX7 may be subject to rapid degradation)

• Buffer optimization based on subcellular localization:

  • Nuclear fraction: Include nuclear lysis buffers with higher salt concentration

  • Cytoplasmic fraction: Use gentler extraction conditions

• Consider tissue-specific extraction protocols as GA2OX7 expression varies between different plant tissues

• Implement protein phosphatase inhibitors if studying post-translational modifications, as GA2OX7 activity may be regulated by phosphorylation

How can GA2OX7 antibodies be used to study hormone crosstalk in plants?

GA2OX7 antibodies provide valuable tools for investigating the complex crosstalk between brassinosteroid and gibberellin signaling pathways:

• Protein level analysis: Quantify changes in GA2OX7 protein abundance following brassinosteroid treatment using Western blot analysis

• Chromatin immunoprecipitation (ChIP): Examine binding of BR-regulated transcription factors like CESTA (CES) to the GA2OX7 promoter

• Co-immunoprecipitation: Identify protein interactions between GA2OX7 and components of hormone signaling pathways

• Subcellular localization studies: Track changes in GA2OX7 distribution between nucleus and cytoplasm in response to hormonal treatments

Research has demonstrated that the basic helix-loop-helix transcription factor CESTA and its homologues BRASSINOSTEROID-ENHANCED EXPRESSION (BEE) 1 and 3 contribute to brassinosteroid-mediated regulation of GA2OX7 . Antibodies enable researchers to visualize these regulatory networks at the protein level.

What methodological approaches can be used to study GA2OX7 in plant stress responses?

For studying GA2OX7's role in stress responses, researchers should:

• Design time-course experiments to capture dynamic changes in GA2OX7 protein levels during stress imposition

• Compare protein abundance with enzyme activity assays to understand functional regulation

• Implement immunolocalization to track changes in subcellular distribution during stress exposure

• Combine with mutant analysis (ces/bee mutants) to correlate protein levels with physiological outcomes

• Use antibodies in conjunction with metabolite analysis (GA110 levels) to establish relationships between enzyme abundance and gibberellin catabolism

GA2OX7 expression is induced by osmotic stress, touch, and abscisic acid treatment , making antibody-based detection particularly valuable for stress physiology research.

How can researchers distinguish between GA2OX7 and other GA2OX family members?

Distinguishing between GA2OX family members requires:

• Selection of antibodies raised against unique epitopes in GA2OX7

• Validation using recombinant proteins of different GA2OX family members

• Complementary approaches combining antibody detection with transcript analysis

• Use of ga2ox7 knockout/knockdown plants as negative controls

• Mass spectrometry validation of immunoprecipitated proteins

This distinction is crucial as different GA2OX classes have distinct substrate preferences: class III GA2OXs (including GA2OX7) catabolize C20-GAs whereas classes I and II directly convert C19-GAs .

What are common challenges in Western blot analysis with GA2OX7 antibodies?

Researchers frequently encounter these challenges when performing Western blots:

• Non-specific binding - Optimize blocking conditions (5% non-fat milk or BSA) and include appropriate controls

• Weak signal detection - Consider:

  • Enrichment strategies (immunoprecipitation)

  • Enhanced chemiluminescence substrates

  • Longer exposure times

• Multiple bands - May indicate:

  • Post-translational modifications

  • Proteolytic degradation

  • Cross-reactivity with homologous proteins

• Inconsistent results - Address through:

  • Standardized sample preparation

  • Careful quantification of total protein loaded

  • Use of internal loading controls (actin, tubulin)

How can immunohistochemistry protocols be optimized for GA2OX7 localization?

For successful immunohistochemical detection of GA2OX7:

• Fixation optimization:

  • 4% paraformaldehyde for 1-4 hours (depending on tissue thickness)

  • Consider ethanol-acetic acid fixation for tissues with high phenolic content

• Antigen retrieval:

  • Citrate buffer (pH 6.0) heat-induced retrieval

  • Enzymatic retrieval using proteinase K for certain tissues

• Background reduction:

  • Block with 3-5% BSA or normal serum from secondary antibody species

  • Include 0.1-0.3% Triton X-100 for improved permeabilization

• Detection system selection:

  • Fluorescent secondary antibodies for co-localization studies

  • Enzyme-based detection (HRP/DAB) for permanent preparations

• Controls:

  • Pre-immune serum

  • Peptide competition

  • Secondary antibody only

What strategies improve reproducibility in GA2OX7 antibody-based research?

To ensure reproducible results:

• Standardize sample collection:

  • Harvest at consistent developmental stages

  • Control environmental conditions (light, temperature, humidity)

  • Document growth parameters precisely

• Implement rigid technical protocols:

  • Use consistent protein extraction buffers

  • Standardize antibody dilutions and incubation times

  • Document lot numbers of antibodies used

• Include comprehensive controls:

  • Positive and negative biological controls

  • Technical controls for antibody specificity

• Quantitative analysis:

  • Use digital image analysis software

  • Normalize to appropriate loading controls

  • Perform statistical analysis across biological replicates

How can GA2OX7 antibody data be integrated with gene expression data?

Integrating protein and transcript data provides comprehensive insights:

• Perform parallel analyses of GA2OX7 protein (Western blot) and transcript levels (qRT-PCR) from the same samples

• Design time-course experiments to identify potential delays between transcriptional and translational responses

• Analyze correlation coefficients between protein and transcript levels across different experimental conditions

• Examine discrepancies that may indicate post-transcriptional regulation mechanisms

• Create integrated models incorporating:

  • Transcriptional regulation (driven by factors like CESTA)

  • Post-translational modifications

  • Protein stability and turnover

For example, research has shown that CES directly binds to the GA2OX7 promoter and is activated by brassinosteroids, but can also act by BR-independent means . Antibody-based approaches can reveal whether these transcriptional changes translate to altered protein abundance.

What experimental designs best capture GA2OX7 dynamics in hormone signaling?

Optimal experimental designs include:

• Hormone treatment time-courses:

  • Short-term (minutes to hours) for immediate responses

  • Long-term (days) for developmental outcomes

• Dose-response studies:

  • Physiological concentration ranges of brassinosteroids

  • Combined hormone treatments to assess crosstalk

• Genetic background comparisons:

  • Wild-type vs. hormone signaling mutants

  • GA2OX7 overexpression lines

• Stress response protocols:

  • Controlled stress imposition

  • Recovery phase monitoring

Research indicates that in plants overexpressing CES, GA2OX7 expression is hyper-responsive to brassinosteroids, GA110 levels are elevated, and amounts of bioactive GA are reduced . Antibody-based approaches can validate these findings at the protein level.

How can researchers validate GA2OX7 antibody specificity for conclusive research?

Comprehensive validation requires:

• Sequence analysis:

  • Compare immunogen sequence with target species GA2OX7

  • Identify potential cross-reactive regions with other GA2OX family members

• Expression system controls:

  • Recombinant GA2OX7 protein expression

  • Heterologous expression in systems like E. coli or yeast

• Genetic approach:

  • CRISPR/Cas9 or T-DNA insertion mutants as negative controls

  • Complementation lines for specificity confirmation

• Advanced analytical techniques:

  • Mass spectrometry of immunoprecipitated proteins

  • Peptide mapping of recognized epitopes

How might GA2OX7 antibodies contribute to understanding climate resilience in crops?

GA2OX7 antibodies can advance climate resilience research through:

• Comparative studies across stress-tolerant and susceptible varieties

• Analysis of GA2OX7 protein dynamics during:

  • Drought stress responses

  • Salt stress adaptation

  • Temperature stress resilience

• Screening of natural variation in GA2OX7 protein abundance and localization

• Evaluation of transgenic approaches targeting GA2OX7 for improved stress tolerance

Research has shown that GA2OX7 acts in the restriction of root elongation under salt stress , suggesting its importance in stress adaptation mechanisms.

What new technologies might enhance GA2OX7 antibody applications?

Emerging technologies with potential applications include:

• Proximity ligation assays to visualize protein-protein interactions in situ

• Super-resolution microscopy for precise subcellular localization

• Automated Western blot systems for higher throughput analysis

• Single-cell proteomics to understand cell-specific GA2OX7 regulation

• CRISPR-based tagging for endogenous GA2OX7 visualization

These approaches will enable researchers to move beyond traditional antibody applications toward more sophisticated understanding of GA2OX7 function in plant development and stress responses.