MTERF1 Antibody

Basic Research Questions

• How to validate MTERF1 antibody specificity for mitochondrial protein studies?

  • Perform knockout validation using CRISPR/Cas9-edited cell lines (e.g., HCT116 or HT29 CRC cells) to confirm antibody signal loss in MTERF1-deficient models .

  • Combine subcellular fractionation (mitochondrial isolation) with Western blotting to verify mitochondrial localization .

  • Use peptide competition assays with the immunogen sequence (e.g., aa 1-200 of human MTERF1) to test binding specificity .

• What experimental models are suitable for studying MTERF1's role in mitochondrial transcription?

  • In vitro transcription assays: Use templates containing LSP (light-strand promoter) or HSP (heavy-strand promoter) with recombinant MTERF1 to assess termination efficiency .

  • Xenograft tumor models: Compare MTERF1-overexpressing vs. knockdown CRC cell lines (e.g., HT29 and HCT116) to evaluate tumor growth and mitochondrial OXPHOS activity .

Advanced Research Questions

• How to resolve contradictions in MTERF1's reported roles in transcription termination vs. replication fork regulation?

  • Mechanistic dissection:

    • Use rolling-circle replication assays to test replication fork stalling at MTERF1-binding sites (reverse orientation shows stronger replication blockage) .

    • Perform strand-specific RT-qPCR to quantify antisense RNA levels in MTERF1 knockout vs. wild-type models .

  • Pathway crosstalk analysis: Investigate AMPK/mTOR signaling modulation via Western blot (p-AMPK/AMPK ratio) in MTERF1-manipulated CRC cells .

• What methodologies detect MTERF1's impact on mitochondrial genome stability?

  • mtDNA copy number quantification: Use qPCR with primers for ND1 (mtDNA) vs. 18S rRNA (nuclear DNA) .

  • Oxidative stress assays: Measure ROS levels (DCFDA staining) and mitochondrial membrane potential (JC-1 ΔΨm assay) in MTERF1-overexpressing cells .

Technical Optimization Challenges

• How to address nonspecific binding in MTERF1 immunoprecipitation (IP) studies?

  • Optimize crosslinking conditions (e.g., formaldehyde fixation) to stabilize transient protein-DNA interactions .

  • Include TEFM protein in IP buffers to mitigate interference with transcription elongation complexes .

Table 1: Validated Applications of MTERF1 Antibodies in Peer-Reviewed Studies

Controversy Navigation

• Why do some studies report MTERF1 as oncogenic while others emphasize housekeeping roles?

  • Context-dependent analysis:

    • In CRC, MTERF1 promotes G1/S transition via AMPK/mTOR, driving proliferation .

    • In non-cancer models, MTERF1 maintains mtRNA stoichiometry by blocking antisense transcription .

  • Experimental design: Compare cell-type-specific interactomes using BioID or APEX2 proximity labeling.

Multi-Omics Integration

• How to link MTERF1-mediated mitochondrial dysfunction to transcriptomic changes?

  • Perform mitochondrial RNA-seq paired with ATAC-seq to map chromatin accessibility changes in MTERF1-KO models .

  • Use Seahorse XF analysis to correlate OCR (oxygen consumption rate) with RNA expression clusters .