MTERF5 Antibody

FAQs for MTERF5 Antibody in Academic Research
Below is a structured collection of research-focused FAQs addressing experimental design, methodological challenges, and advanced applications of MTERF5 antibodies. Data are derived from peer-reviewed studies, with emphasis on functional characterization and validation protocols.

How to validate the specificity of MTERF5 antibodies in mitochondrial studies?

• Methodological Answer:

  • Western Blot: Use mitochondrial lysates from wild-type and MTERF5 knock-down/knock-out models (e.g., Drosophila D.Mel-2 cells). A specific antibody should show reduced or absent bands in knock-down samples .

  • Immunocytochemistry: Co-stain with mitochondrial markers (e.g., MitoTracker) to confirm subcellular localization. In Drosophila, FLAG-tagged D-MTERF5 showed perfect overlap with mitochondrial networks .

  • Negative Controls: Include tissues/cells lacking MTERF5 expression (e.g., non-insect models) to rule off-target binding.

What experimental models are optimal for studying MTERF5 function?

• Methodological Answer:

  • Insect-Specific Systems: Use Drosophila melanogaster cell lines (e.g., D.Mel-2) or transgenic flies, as MTERF5 is absent in vertebrates .

  • Knock-Down Approaches: Employ dsRNA targeting MTERF5 coding sequences (e.g., 787 bp fragment) to reduce transcript levels by >90% (validated via RT-PCR) .

  • Mitochondrial Isolation: Purify mitochondria via differential centrifugation for protein-DNA interaction assays (e.g., EMSA, DNase footprinting) .

How does MTERF5 influence mitochondrial transcription?

• Key Findings:

  • MTERF5 depletion in Drosophila increases upstream transcripts (e.g., ND2, COI) by 30–50% and decreases downstream transcripts (e.g., cyt b, ND4/4L) by 40–50% .

  • Acts antagonistically to DmTTF (transcription termination factor), modulating POLRMT progression (Fig. 2B ).

Table 1: Transcript Level Changes in D-MTERF5 Knock-down

How to resolve contradictory data on MTERF5’s role in transcription termination?

• Methodological Answer:

  • Multi-Approach Validation: Combine EMSA, DNase footprinting, and transcriptional profiling. For example, EMSA revealed D-MTERF5 requires DmTTF to bind mtDNA (Fig. 3B ).

  • Homology Modeling: Predict protein-protein interaction domains (e.g., SWISS-MODEL) to explain cooperative binding with DmTTF .

  • Co-IP Assays: Verify physical interactions using His/FLAG-tagged proteins in bacterial/pulldown systems .

What mechanisms underlie MTERF5-DmTTF functional antagonism?

• Key Insights:

  • Bidirectional Modulation: D-MTERF5 extends DmTTF’s DNase footprint by 10–15 nucleotides, altering POLRMT arrest (Fig. 3D ).

  • Stoichiometric Balance: Co-regulation by DREF ensures coordinated expression of MTERF5 and DmTTF for transcription fine-tuning .

Table 2: Functional Comparison of MTERF5 and DmTTF

How to design conditional knock-down experiments for MTERF5?

• Methodological Answer:

  • Temporal Control: Use inducible RNAi systems (e.g., tetracycline-regulated promoters) to avoid developmental lethality.

  • Transcript Monitoring: Perform qRT-PCR at multiple timepoints to correlate MTERF5 levels with mitochondrial RNA changes (e.g., ND2, cyt b) .

  • Rescue Experiments: Overexpress wild-type MTERF5 in knock-down models to confirm phenotype reversibility.