SAC3B Antibody

Here’s a curated collection of FAQs for researchers working with SAC3B antibody in academic contexts, synthesized from peer-reviewed studies and technical data:

What are the primary applications of SAC3B antibody in plant epigenetic studies?

SAC3B antibody is critical for investigating its role in the TREX-2 complex, which links mRNA export to transcriptional anti-silencing. Key applications include:

• Chromatin Immunoprecipitation (ChIP): Detecting SAC3B-associated histone modifications (e.g., H3K9me2 elevation) at silenced loci .

• Protein interaction assays: Co-immunoprecipitation (Co-IP) with partners like THP1 and NUA to validate TREX-2 complex integrity .

• Subcellular localization: Confocal microscopy to assess SAC3B’s nuclear pore association and mRNA export defects in mutants .

Key validation data:

How to validate SAC3B antibody specificity in Arabidopsis?

• Knockout controls: Compare signal intensity in sac3b mutants (e.g., p31 allele) vs. wild-type .

• Cross-reactivity tests: Use Western blot on extracts from SAC3B overexpression lines to confirm band size (~120 kDa) .

• Functional rescue: Reintroduce SAC3B-MYC/FLAG-tagged constructs and verify antibody recognition .

How to resolve contradictions in SAC3B’s role in siRNA vs. DNA methylation regulation?

Genome-wide studies show SAC3B primarily affects siRNA levels at TAS loci (e.g., 22-nt siRNA reduction by 40%) but has minor DNA methylation effects (<5% change genome-wide). Methodological considerations:

• Stratified analysis: Separate heterochromatin (e.g., transposons) from euchromatin (e.g., TAS3) using bisulfite-seq .

• Time-course experiments: Track siRNA dynamics during developmental stages to distinguish direct vs. indirect effects .

Conflict resolution workflow:

• Confirm siRNA quantification via smRNA-seq with spike-in controls.

• Pair DNA methylation data with ROS1 demethylase activity assays .

What experimental designs clarify SAC3B’s interplay with DNA repair pathways in viral resistance?

atxr5 atxr6 mutants with SAC3B dysfunction show geminivirus resistance via RAD51 retention. Key approaches:

• Dual ChIP-seq: Profile RAD51/SAC3B co-occupancy at viral DNA integration sites .

• Viral load quantification: Compare qPCR-based viral DNA levels in sac3b atxr5 atxr6 vs. single mutants .

Critical data table:

How to differentiate R-loop-dependent vs. independent gene silencing in SAC3B studies?

Despite R-loop detection in sac3b, silencing is R-loop-independent. Experimental strategies:

• DRIP-seq with S9.6 antibody: Compare R-loop levels at silenced loci (e.g., d35S::LUC) in sac3b vs. ros1 .

• RNase H1 overexpression: Test if R-loop dissolution rescues silencing (no effect observed in ).

Methodological Notes for Robust Results

• Antibody storage: Lyophilized SAC3B antibody requires reconstitution in PBS + 0.1% BSA; avoid freeze-thaw cycles .

• ChIP optimization: Use 12-day-old seedlings and anti-Pol II CTD (8WG16) for occupancy studies .