At2g29380 Antibody

The Arabidopsis thaliana gene At2g29380 encodes HAI3 (Highly ABA-Induced PP2C Gene 3), a protein phosphatase 2C involved in abscisic acid signaling. Below are research-focused FAQs addressing technical considerations for working with At2g29380 antibodies, organized by experimental complexity and supported by methodological details from peer-reviewed protocols and genomic studies 4.

What transient expression systems optimize At2g29380 antibody performance in TF-target studies?

The TARGET (Transient Assay Reporting Genome-wide Effects of Transcription Factors) system provides a 14-day protocol :

• Protoplast preparation:

  • Use 4-week-old Arabidopsis rosette leaves

  • Enzyme solution: 1.5% Cellulase R10 + 0.4% Macerozyme R10

• GR-bZIP1 fusion construct:

  • pTARGET vector with glucocorticoid receptor (GR) domain

  • Dexamethasone induction (10 μM, 6h pulse)

• Antibody application:

  • ChIP-seq: Anti-GR (1:500) + Protein A/G magnetic beads

  • Co-staining with nuclear marker DAPI (1 μg/mL)

Key parameters:

How to resolve discrepancies in At2g29380-binding data across stress conditions?

Multivariate analysis framework:

• Experimental variables:

  • ABA concentration gradient (0, 10, 100 μM)

  • Time-course sampling (0, 2, 6, 24h)

• Technical controls:

  • Cycloheximide (50 μg/mL) to block new protein synthesis

  • Parallel RIP-seq with anti-HAI3 vs IgG isotype

• Data normalization:

  • Spike-in Arabidopsis RNA (External RNA Controls Consortium mix)

  • Reference genes: PP2AA3 (AT1G13320), UBC21 (AT5G25760)

Common artifact sources:

• Variable PP2C activity in extraction buffers (include 20 mM NaF)

• Light-dependent phosphorylation states (harvest at ZT4)

What orthogonal methods confirm At2g29380-antibody interaction specificity in vivo?

Advanced validation pipeline:

Troubleshooting:

• For low FRET efficiency: Use phosphatase inactive mutant (D64A)

• Include Mg²⁺ chelators (10 mM EDTA) in binding assays

How to design genome-editing controls for At2g29380 functional studies?

CRISPR-Cas9 strategy:

• Dual sgRNA design:

  • Target exons 2 & 4 (sgRNA1: 5'-GCACCTGCAGTACGTGCTCG-3')

  • Avoid off-targets in At2g29390 (3 mismatches minimum)

• Phenotypic validation:

  • Stomatal aperture assay (ABA 10 μM vs control)

  • Ion leakage after drought stress (30% PEG 6000)

Expected physiological data: