MORC6 Antibody

How to validate MORC6 antibody specificity in plant chromatin studies?

Methodological approach:

• Perform Western blot using Arabidopsis thaliana wild-type and morc6 knockout mutants. Validate absence of signal in mutants .

• Use immunofluorescence with nuclear markers (e.g., histone H3) to confirm subcellular localization in root tip cells .

• Conduct peptide competition assays by pre-incubating the antibody with the immunogen peptide; signal reduction confirms specificity .

Key findings:

• MORC6 colocalizes with RdDM sites and Pol V .

• Loss of signal in morc6 mutants confirms antibody reliability .

What experimental designs are optimal for studying MORC6’s role in RdDM-dependent vs. RdDM-independent pathways?

Methodological approach:

• Genetic crosses: Combine morc6 mutants with mom1 or pol V mutants to isolate RdDM-dependent effects .

• ChIP-seq: Compare MORC6 binding sites in wild-type and RdDM mutants (e.g., nrpe1) to identify RdDM-independent targets .

• ATAC-seq: Assess chromatin accessibility changes in morc6 mutants to distinguish chromatin compaction roles .

Key findings:

• MORC6 interacts with PIAL2 in the MOM1 complex for RdDM-dependent silencing .

• RdDM-independent functions involve chromatin compaction at stress-responsive loci .

How to resolve contradictions in MORC6’s role in DNA methylation maintenance?

Methodological approach:

• Epistasis analysis: Compare DNA methylation levels in morc6, mom1, and double mutants using whole-genome bisulfite sequencing .

• Time-course experiments: Track de novo methylation at RdDM targets (e.g., FWA) after ZF-MORC6 tethering in mutant backgrounds .

Key findings:

• MORC6 acts downstream of MOM1 but upstream of Pol V in RdDM .

• Partial redundancy with other MORC proteins explains locus-specific methylation defects .

What functional assays confirm MORC6’s interaction with chromatin remodelers?

Methodological approach:

• Yeast Two-Hybrid (Y2H): Test MORC6’s GHKL domain against PIAL2 or ACD proteins .

• Co-IP/MS: Immunoprecipitate MORC6-FLAG from transgenic plants and identify co-purifying partners (e.g., MBD–ACD complex) .

• In vitro ATPase assays: Measure ATP hydrolysis activity of recombinant MORC6 to confirm enzymatic function .

Key findings:

• MORC6’s GHKL domain mediates interaction with ACD15.5/21.4 .

• ATPase activity is critical for chromatin compaction .

How to investigate MORC6’s role in human disease models?

Methodological approach:

• CRISPR knockouts: Generate MORC6-deficient cell lines to assess transcriptional dysregulation (e.g., rDNA silencing) .

• ChIP-qPCR: Profile MORC6 binding at disease loci (e.g., VAR1 in cancer) under stress conditions .

• Transcriptome analysis: Compare RNA-seq data from patient samples with MORC6 mutations vs. wild-type .

Key findings:

• MORC6 depletion disrupts 45S rDNA silencing via MBD–ACD recruitment .

• MORC6 mutations correlate with autoimmune disorders (e.g., dermatomyositis) .

What controls are essential when using MORC6 antibodies in co-IP experiments?

Methodological approach:

• Negative controls: Use IgG from pre-immune serum and morc6 mutant lysates .

• Competition controls: Include immunogen peptide in parallel IPs .

• Crosslinking optimization: Test formaldehyde vs. DSG crosslinkers for chromatin-bound complexes .

Advanced Research Focus: Integrating MORC6 Data with Multi-Omics

Methodological approach:

Key findings:

• MORC6 loss increases DNA accessibility for stress-responsive TFs .

• Stochastic silencing in morc6 mutants suggests backup silencing mechanisms .

Addressing Technical Limitations in MORC6 Studies

Methodological approach:

• Multiplexed IF-FISH: Combine immunofluorescence with FISH to correlate MORC6 localization with target loci (e.g., FWA) .

• Crispr-mediated epitope tagging: Endogenously tag MORC6 for native ChIP-seq .

• Structure-function analysis: Express MORC6 truncations (e.g., ΔGHKL) to dissect domain roles .

Key findings:

• MORC6’s GHKL domain is essential for ACD protein interactions .

• Truncations disrupt RdDM but not chromatin compaction .