GA20OX2 Antibody

Here’s a structured collection of FAQs for researchers working with GA20OX2 antibodies, based on experimental methodologies and findings from recent studies:

How do I validate the specificity of GA20OX2 antibodies in plant tissue samples?

• Methodology:

  • Perform Western blotting using protein extracts from Arabidopsis thaliana wild-type and ga20ox2 knockout mutants. A band at ~45 kDa (predicted molecular weight) should appear only in wild-type samples .

  • Use immunolocalization in hypocotyls or stems (high GA20OX2 expression sites) with GFP-tagged controls (e.g., 35S::ATHB2-GFP lines) to confirm subcellular localization .

  • Cross-reactivity testing: Include Brassica species (e.g., Brassica rapa) due to conserved epitopes .

What experimental designs are optimal for studying GA20OX2 transcriptional regulation?

• Approach:

  • Promoter-reporter assays: Fuse GA20OX2 promoter fragments (e.g., 1.5-kb, 1.0-kb, 0.5-kb upstream regions) to luciferase and co-transform with effectors like 35S::ATHB2-GFP or 35S::TCP13-GFP in protoplasts .

  • Key controls: Include Renilla luciferase for normalization and mutagenized promoter variants (e.g., ABS1/ABS2 mutants) to identify binding sites .

How do I resolve contradictory data in GA20OX2 expression under shade conditions?

• Troubleshooting:

  • Check temporal dynamics: ATHB2 induction precedes GA20OX2 activation; sample tissues at multiple time points post-shade exposure .

  • Assess TCP13 levels: Overexpression of TCP13 represses GA20OX2, while ATHB2 co-expression reverses this effect .

How does the GAF1-DELLA-TPR complex regulate GA20OX2 feedback?

• Mechanistic insights:

  • ChIP-qPCR: Use anti-GAF1 or anti-TPR4 antibodies to confirm binding to the GA20OX2 promoter (−1030 to −1011 region) .

  • GA3 treatment: Apply 10 μM GA3 to ga1-3 mutants expressing ΔPAM; observe repression of GA20OX2 activation .

What strategies identify GA20OX2-interacting transcription factors in vivo?

• Workflow:

  • Yeast one-hybrid screening: Screen a cDNA library with the GA20OX2 promoter (−1.5-kb region) to isolate binding TFs like ATHB2 .

  • EMSA: Use biotin-labeled ABS1/ABS2 oligonucleotides and purified His-tagged ATHB2 to validate direct binding .

How to quantify GA20OX2 protein dynamics during hypocotyl elongation?

• Quantitative approach:

  • ELISA/Western blot: Use the GA20OX2 antibody with protein extracts from shaded vs. control seedlings. Normalize to total protein (e.g., Coomassie staining) .

  • Mutant validation: Compare elongation rates in ga20ox2 mutants vs. wild-type under shade .

Data Integration Table

Methodological Notes

• Antibody storage: Lyophilized GA20OX2 antibodies require storage at -20°C with manual defrosting .

• Cross-species use: Validated in Arabidopsis, Brassica rapa, and B. napus .