At1g79990 Antibody

FAQs for At1g79990 Antibody in Academic Research

How to validate specificity of At1g79990 antibody in plant tissue experiments?

• Methodological Answer:

  • Step 1: Perform Western blot using protein extracts from wild-type and At1g79990 knockout mutants. A specific antibody will show a band in wild-type but not in mutants .

  • Step 2: Use immunofluorescence in transgenic plants expressing tagged At1g79990 (e.g., GFP fusion) to confirm colocalization .

  • Step 3: Cross-validate with orthogonal methods like ELISA or immunoprecipitation followed by mass spectrometry .

  Common Pitfalls:

  • Non-specific binding due to epitope similarity with homologous proteins (e.g., other bZIP transcription factors). Pre-adsorb the antibody with recombinant proteins from closely related family members .

What protocols optimize At1g79990 antibody performance in ChIP-seq for nitrogen-responsive transcription factors?

• Methodological Answer:

  • Fixation: Use formaldehyde crosslinking (1% v/v, 10 min) followed by glycine quenching to preserve DNA-protein interactions .

  • Sonication: Fragment chromatin to 200–500 bp for high-resolution binding site detection (e.g., Covaris S220, 20% duty cycle, 6 cycles).

  • Antibody Dilution: Titrate antibody (1:50–1:200) using positive controls (e.g., known At1g79990 target promoters like ASN1 or GLN1.3) .

  Validation:

  • Include a no-antibody control and spike-in synthetic DNA standards to normalize technical variability .

How to resolve contradictory data between At1g79990 antibody-based ChIP-seq and RNA-seq in nitrogen signaling studies?

• Methodological Answer:

  • Scenario 1: ChIP-seq peaks without corresponding RNA-seq changes.

    • Investigate post-translational modifications (e.g., phosphorylation) affecting At1g79990 activity .

    • Use transient transfection assays to test if DNA binding correlates with transcriptional activation .

  • Scenario 2: RNA-seq changes without ChIP-seq peaks.

    • Check for indirect regulation via intermediary TFs (e.g., CCA1 or GLK1) using co-immunoprecipitation .

  Tools:

  • Integrate ATAC-seq to assess chromatin accessibility at putative binding sites .

Can At1g79990 antibody cross-react with orthologs in crops like maize or rice?

• Experimental Design:

  • Step 1: Perform phylogenetic analysis to identify conserved epitopes (e.g., bZIP DNA-binding domain).

  • Step 2: Test cross-reactivity via Western blot using protein extracts from maize (ZmbZIP22) or rice (OsbZIP48) .

  • Step 3: Validate functional relevance by silencing orthologs and assessing rescue with At1g79990 overexpression .

  Key Finding:

  • Limited cross-reactivity observed in monocots due to divergent N-terminal domains .

How to combine At1g79990 antibody with multi-omics for network-level analysis of nitrogen assimilation?

• Workflow:

  • ChIP-seq: Identify direct targets of At1g79990 under low/high nitrogen .

  • Metabolomics: Quantify glutamine/glutamate ratios via LC-MS to link TF activity to N-status .

  • Machine Learning: Train models (e.g., Random Forest) to predict regulatory nodes using binding sites and metabolite levels .

  Integration Table:

  Technique	Purpose	Key Parameter
  ChIP-seq	DNA binding	Peak fold-change ≥ 2 (FDR < 0.05)
  RNA-seq	Transcriptional output	TPM ≥ 1 in ≥ 50% samples
  Metabolomics	N-metabolite flux	Gln/Glu ratio ± 20% from baseline

Methodological Considerations from Literature

• Antibody Design: Ensure CDR loops are optimized for epitope accessibility using tools like RosettaAntibodyDesign .

• Troubleshooting: For low signal in immunofluorescence, enhance permeability with 0.1% Triton X-100 and protease inhibitors .

• Data Reproducibility: Use biological triplicates and include At1g79990 overexpression lines as positive controls .