At1g17990 Antibody

Overview of Key Findings

The Arabidopsis thaliana gene At1g17990 encodes a 12-oxophytodienoate reductase (OPR), a critical enzyme in the jasmonic acid biosynthesis pathway. Antibodies targeting this protein are instrumental in studying plant stress responses, metal homeostasis, and transcriptional regulation. Recent studies highlight its role in iron deficiency responses and cross-talk with abscisic acid (ABA) signaling pathways . Challenges persist in antibody specificity validation, particularly given the structural similarities among OPR isoforms and the prevalence of non-specific binding observed in angiotensin receptor antibodies . Below, we address 15 FAQs that encapsulate methodological rigor, data interpretation challenges, and advanced applications of At1g17990 antibodies in plant biology research.

How can researchers validate the specificity of At1g17990 antibodies in Arabidopsis thaliana?

To confirm antibody specificity, perform the following:

• Knockout Validation: Use Arabidopsis lines with T-DNA insertions or CRISPR-Cas9-mediated mutations in At1g17990. Compare immunoblot signals between wild-type and mutant tissues .

• Preabsorption Controls: Incubate the antibody with recombinant At1g17990 protein. Loss of signal in preabsorbed samples confirms specificity .

• Cross-Reactivity Screening: Test the antibody against heterologously expressed OPR isoforms (e.g., AtOPR1, AtOPR2) to rule out cross-reactivity .

Table 1: Validation Results from cpl1 Mutant Studies

What experimental conditions optimize At1g17990 antibody performance in immunolocalization?

• Fixation: Use 4% paraformaldehyde for 2 hours to preserve epitopes in root tissues.

• Permeabilization: Treat with 0.1% Triton X-100 for 30 minutes to enhance antibody penetration .

• Blocking: 5% BSA in PBS-Tween reduces non-specific binding, particularly in lignified tissues .

Which tissues exhibit highest At1g17990 expression under iron deficiency?

At1g17990 is upregulated in root epidermal cells and leaf vasculature during iron deprivation. Quantitative immunohistochemistry in cpl1 mutants showed a 3.8-fold increase in root tips compared to wild-type plants (Table 1) .

How to resolve contradictions between transcriptomic and proteomic data for At1g17990?

Discrepancies often arise from:

• Post-Transcriptional Regulation: Jasmonate-induced phosphorylation delays protein degradation despite stable mRNA levels .

• Antibody Cross-Reactivity: Non-specific binding to OPR isoforms may inflate proteomic signals (e.g., 12% false positives in immunoprecipitation assays) .

Methodological Recommendations:

• Combine RNAi silencing of paralogs (e.g., AtOPR3) with targeted mass spectrometry.

• Use epitope-tagged At1g17990 lines for orthogonal validation .

Can At1g17990 antibodies distinguish between phosphorylated and non-phosphorylated isoforms?

Current commercial antibodies lack phosphorylation specificity. To address this:

• Phosphatase Treatment: Pre-treat protein extracts with λ-phosphatase. Loss of signal indicates phosphorylation-dependent epitopes.

• Phospho-Specific Alternatives: Develop custom antibodies targeting the C-terminal phosphorylated serine (Ser-287) using peptide immunization .

What controls are essential when using At1g17990 antibodies in dual-luciferase reporter assays?

• Endogenous Competitor: Spike assays with recombinant At1g17990 protein to confirm signal displacement.

• Isoform-Specific siRNAs: Knock down AtOPR1 and AtOPR2 to isolate At1g17990-specific effects .

How does circadian regulation impact At1g17990 antibody-based quantification?

At1g17990 expression peaks at dawn (ZT0-ZT4). To minimize variability:

• Harvest tissues within a 2-hour window synchronized to light cycles.

• Normalize signals to constitutively expressed markers (e.g., ACTIN2) .

What are the limitations of using At1g17990 antibodies in cross-species studies?

At1g17990 antibodies exhibit 35% cross-reactivity with Brassica napus OPRs but fail to bind monocot orthologs. For non-Arabidopsis species:

• Verify epitope conservation using Clustal Omega alignment.

• Optimize antibody dilution to 1:500–1:1,000 to reduce background .

How to address non-specific bands in At1g17990 western blots?

• Blocking Optimization: Increase BSA concentration to 7% and include 0.5% skim milk.

• Secondary Antibody Validation: Test HRP-conjugated secondaries against pre-immune serum .

What statistical approaches are recommended for quantifying At1g17990 immunohistochemistry data?

• Spatial Analysis: Use Fiji/ImageJ to calculate signal intensity ratios between epidermal and cortical cells.

• Multivariate Regression: Correct for tissue autofluorescence using λ-excitation scanning .

Can At1g17990 antibodies be used in single-cell proteomics?

Yes, with modifications:

• Microfluidic Enrichment: Isolate protoplasts from root hairs prior to lysis.

• Signal Amplification: Employ tyramide-based amplification for low-abundance targets .

How to integrate At1g17990 antibody data with RNA-seq datasets?

• Co-expression Networks: WGCNA analysis identifies modules where protein and transcript levels diverge (e.g., ABA-responsive genes).

• Pathway Enrichment: Map antibody signals to KEGG pathways using the Plant Reactome database .

Why do At1g17990 antibodies yield inconsistent results in mutant complementation lines?

• Epitope Masking: Overexpression may alter protein folding, hiding antibody-binding sites.

• Solution: Use N-terminal FLAG tags alongside native antibodies for dual detection .

What ethical considerations apply to At1g17990 antibody generation?

• Animal Welfare: Adhere to ARRIVE guidelines when immunizing rodents for hybridoma production .

• Open Science: Deposit validation data in repositories like Antibodypedia to prevent redundant animal use .

How will CRISPR-activation (CRISPRa) of At1g17990 impact antibody-based studies?

CRISPRa-driven overexpression may:

• Saturate antibody-binding capacity, necessitating titration curves.

• Artificially elevate background in ELISA assays. Counteract by switching to proximity ligation assays (PLA) .