GBF2 Antibody

Basic Research Questions

• How to validate GBF2 antibody specificity in plant stress-response studies?

  • Perform immunoblotting using plant protein extracts from wild-type and GBF2 knockout mutants. A valid antibody will show a band only in wild-type samples .

  • Include peptide competition assays: pre-incubate the antibody with excess GBF2-derived peptide to confirm signal loss .

  • For transcriptional studies, combine with cycloheximide treatment (10 µg/mL, 4-hour pre-incubation) to isolate direct transcriptional targets by inhibiting new protein synthesis .

• What are optimal conditions for GBF2 antibody in chromatin immunoprecipitation (ChIP)?

  • Use crosslinking buffer (1% formaldehyde, 10 min) followed by nuclear extraction and sonication (15 cycles, 30 sec ON/OFF) .

  • Validate enrichment via qPCR with primers targeting known GBF2-regulated promoters (e.g., stress-response genes like GTP-binding protein 107) .

Advanced Experimental Design

• How to resolve cross-reactivity between GBF2 and homologous transcription factors (e.g., GBF1)?

  • Employ alanine-scanning mutagenesis on the GBF2 epitope to identify critical binding residues .

  • Compare binding kinetics using surface plasmon resonance (SPR) with recombinant GBF1/GBF2 proteins (see table below) :

  Parameter	GBF2 (KD)	GBF1 (KD)
  Kon (M⁻¹s⁻¹)	2.1×10⁵	6.7×10³
  Koff (s⁻¹)	1.8×10⁻³	4.2×10⁻²

• What experimental strategies distinguish direct GBF2-DNA interactions from indirect associations?

  • Combine ChIP-seq with ATAC-seq to correlate binding sites with open chromatin regions .

  • Use dominant-negative GBF2 mutants lacking DNA-binding domains to disrupt native interactions in transient transfection assays .

Data Interpretation & Contradictions

• How to address discrepancies between ELISA and Western blot results for GBF2 detection?

  • Epitope accessibility: ELISA may detect linear epitopes, while Western blots under denaturing conditions could mask conformational epitopes (e.g., CU-28-24 mAb in ).

  • Validate via immunoprecipitation-mass spectrometry (IP-MS) to confirm protein identity in native complexes .

• Why do GBF2 antibody signals vary across stress conditions (e.g., cold vs. salinity)?

  • Post-translational modifications: Phosphorylation or ubiquitination under specific stresses may alter epitope availability .

  • Quantify temporal dynamics using time-course Western blots (0–24 hr post-stress) paired with mRNA-seq to decouple transcriptional vs. translational regulation .

Methodological Optimization

• Which controls are critical for GBF2 immunohistochemistry (IHC) in plant tissues?

  • Include isotype-matched IgG and antigen-preabsorbed antibody controls .

  • Use autofluorescence quenching (0.1% Sudan Black B, 10 min) for fluorescent IHC in chlorophyll-rich tissues .

• How to enhance GBF2 antibody sensitivity in low-abundance scenarios?

  • Implement signal amplification systems (e.g., Tyramide SuperBoost™) for indirect detection .

  • Optimize antigen retrieval (10 mM citrate buffer, pH 6.0, 95°C, 20 min) for formalin-fixed samples .

Cross-Species Reactivity

• Does GBF2 antibody recognize orthologs in non-model plant species?

  • Perform phylogenetic alignment of the epitope region across species (e.g., residues 120-145 in Arabidopsis GBF2).

  • Test reactivity against heterologously expressed proteins in E. coli or N. benthamiana .