tomm-20 Antibody

What experimental controls are essential for validating TOMM20 antibody specificity in western blotting?

• Positive controls: Lysates from mitochondrial-rich tissues (e.g., liver) or cell lines (HeLa, SH-SY5Y) showing a band at ~14–16 kDa .

• Negative controls: Knockout cell lines or siRNA-mediated TOMM20 knockdown samples .

• Secondary antibody controls: Omit primary antibody or use isotype-matched IgGs to rule out non-specific binding .

Key validation data:

How does fixation method impact TOMM20 antibody performance in immunofluorescence?

• Paraformaldehyde (4%): Preserves mitochondrial morphology but requires permeabilization (0.1% Triton X-100) .

• Methanol fixation: Avoid for TOMM20 due to epitope denaturation risks .

• Antigen retrieval: Required for formalin-fixed paraffin-embedded (FFPE) tissues using EDTA buffer (pH 8–9) .

Critical findings:

• TOMM20-APEX2 interactome contains 21 MOM proteins vs. 7 in TOMM70-APEX2 .

• Nuclear/ribosomal proteins in datasets may reflect stress-induced interactions .

What computational tools complement TOMM20 antibody-based studies of mitochondrial import mechanisms?

• Prediction algorithms:

  • MitoFates (transit peptide prediction)

  • STRING-DB (interaction network analysis)

• Quantitative approaches:

  • Label-free quantification (LFQ) for interactome studies

  • Spatial proteomics tools (e.g., MitoCarta3.0)

Case study:

How to correlate TOMM20 expression levels with clinical outcomes using IHC?

• Scoring system:

  • 0: No staining

  • 1+: Weak mitochondrial signal

  • 2+: Moderate granular pattern

  • 3+: Strong diffuse staining

Clinical validation:

Why do TOMM20 antibodies show species-specific performance despite high sequence homology?

• Structural insight: The 4F3 clone recognizes a conformational epitope disrupted in pig homologs .

• Solution:

  • Validate cross-reactivity using peptide arrays

  • Consider recombinant antibodies (e.g., EPR15581-54 clone) for multi-species studies