At5g03810 Antibody

Basic Research Questions

• How to validate At5g03810 antibody specificity for Arabidopsis studies?

  • Method:

    1. Perform Western blot (WB) using recombinant At5g03810 protein (immunogen) and lysates from wild-type vs. At5g03810 knockout mutants .

    2. Use transient overexpression in Nicotiana benthamiana to confirm antibody recognition of epitope-tagged proteins .

    3. Validate cross-reactivity with paralogs (e.g., VviMYB108A/B in grapevine) using competitive ELISA .

  • Critical Data:

    Sample Type	Expected Band Size (kDa)	Observed Result
    Recombinant	~35 (Q9FFN0)	Single band
    WT Arabidopsis	Variable (tissue-specific isoforms)	Tissue-dependent

• What are the optimal applications for At5g03810 antibody in plant molecular biology?

  • Preferred Techniques:

    • Chromatin Immunoprecipitation (ChIP): Identify DNA-binding sites of At5g03810 in floral organ primordia .

    • Co-expression Network Analysis: Pair with WGCNA or tau analysis to map tissue-specific regulative networks (e.g., anther development) .

  • Limitations:

    • Avoid fixed tissues for WB due to epitope masking .

    • Low sensitivity for post-translationally modified isoforms without signal amplification .

• How to troubleshoot non-specific binding in At5g03810 antibody assays?

  • Solutions:

    • Pre-adsorb the antibody with E. coli-expressed At5g03810 protein (1:5 ratio, 1 hr at 4°C) .

    • Include tissue-specific controls (e.g., root vs. flower extracts) to rule out cross-reactivity with unrelated MYB TFs .

Advanced Research Questions

• How to design experiments using At5g03810 antibody to resolve contradictory expression data across studies?

  • Case Example: Discrepancies in reported anther-specific vs. ubiquitous expression .

  • Approach:

    1. Combine DAP-Seq (DNA affinity purification sequencing) with single-cell RNA-seq to distinguish direct DNA binding from indirect transcriptional effects .

    2. Use At5g03810 overexpression lines in Arabidopsis to assess dose-dependent feedback regulation .

  • Data Integration Table:

    Technique	Resolves	Key Insight
    DAP-Seq	DNA-binding vs. expression	Identified 30/47 STS family genes as direct targets
    WGCNA	Tissue-specific networks	Anther-specific HCTs linked to pollen fertility

• What methodologies address At5g03810’s role in both development and stress responses?

  • Experimental Workflow:

    1. Condition-Specific Elicitation: Treat Arabidopsis cell cultures with jasmonic acid or Botrytis cinerea, then perform ChIP-seq .

    2. Metabolite Profiling: Quantify stilbenoid/anthocyanin levels in At5g03810 mutants vs. WT using LC-MS .

  • Key Finding: At5g03810 binds promoters of STS and ROMT genes, linking it to secondary metabolite flux .

• How to integrate At5g03810 antibody data with multi-omics datasets?

  • Pipeline:

    1. Map ChIP-seq peaks to MapMan ontology terms (e.g., "stilbenoid biosynthesis") .

    2. Cross-reference with gene-centered co-expression networks from 1,400+ transcriptomic datasets .

  • Output Example:

    Target Gene	Function	Regulatory Evidence
    STS1	Stilbene synthase	DAP-Seq + co-expression
    ROMT	Resveratrol O-methyltransferase	ChIP + metabolite correlation

Methodological Notes

• Antibody Storage: Aliquot in 50% glycerol/PBS (-80°C) to retain activity >2 years .

• Ethical Compliance: For transgenic lines (e.g., At5g03810-OE in tomato), adhere to national biosafety guidelines .