TFAM Antibody

What is TFAM and why is it an important research target?

TFAM (Transcription Factor A, Mitochondrial) is a key activator of mitochondrial transcription and participates in mitochondrial genome replication. It binds to mitochondrial promoter DNA, aids in transcription of the mitochondrial genome, and helps regulate mitochondrial genome copy number . As a double box High-mobility group DNA-binding and bending protein, TFAM is essential for embryonic development and plays a crucial role in mitochondrial function. Research has shown that TFAM is required for maintaining mitochondrial genome integrity, with disruptions linked to conditions like Kearns-Sayre syndrome and other mitochondrial disorders .

What forms of TFAM antibodies are available for research applications?

TFAM antibodies are available in several forms:

• Monoclonal antibodies (e.g., clone 18G102B2E11)

• Polyclonal antibodies targeting different epitopes

• Species-specific antibodies (human, mouse, rat)

• Region-specific antibodies:

  • N-terminal targeting (e.g., AA 214-241)

  • C-terminal targeting (e.g., AA 227-246)

  • Full-length protein targeting

• Conjugated antibodies (unconjugated, HRP, FITC, biotin, Cy3, DyLight488)

How should I design experiments to study TFAM's role in mitochondrial DNA maintenance?

Design comprehensive experiments that examine both TFAM binding to mtDNA and its functional effects:

• DNA binding assessment: Use chromatin immunoprecipitation (ChIP) with validated TFAM antibodies to analyze TFAM-mtDNA interactions .

• Functional impact: Employ dual approaches:

  • Analyze mtDNA content via qPCR using primers specific for mitochondrial genes (e.g., ND1) normalized to nuclear genes (e.g., S12)

  • Assess mtDNA transcription through RT-PCR or RNA-seq analysis

• Protein interaction studies: Combine co-IP with TFAM antibodies to identify interactions with TFB1M and TFB2M , followed by confirmatory proximity ligation assays.

• Validation controls: Include:

  • TFAM knockout/knockdown controls

  • Species-matched isotype controls

  • Blocking peptide competition assays

  • Secondary antibody-only controls

How can I accurately assess TFAM post-translational modifications in different cellular contexts?

TFAM function is regulated by post-translational modifications (PTMs), which require specialized detection methods:

• PTM-specific detection strategy:

  • Use standard TFAM antibodies for total protein detection

  • Employ phosphorylation-state specific antibodies for phospho-TFAM

  • Use anti-acetyl-lysine antibodies following TFAM immunoprecipitation

• Quantitative assessment:

  • For acetylation stoichiometry analysis, implement isotopic chemical acetylation approaches using deuterium-labeled acetic anhydride followed by LC-MS/MS analysis

  • For phosphorylation analysis, use phospho-enrichment prior to mass spectrometry

• DNA binding impact:

  • Research shows TFAM bound to DNA is less reactive to both enzymatic phosphorylation and nonenzymatic acetylation

  • Use EMSAs to evaluate how PTMs affect TFAM's DNA binding capacity

• Functional consequences:

  • Assess transcription initiation and processivity with in vitro transcription assays

  • Research indicates phosphorylated and acetylated TFAM enhance transcription processivity without affecting initiation

What are common pitfalls in Western blot experiments with TFAM antibodies and how can they be resolved?

Several issues can affect TFAM detection by Western blot:

• Inconsistent molecular weight detection:

  • TFAM has an expected molecular weight of ~24 kDa , but may appear at different sizes due to:

    • Post-translational modifications

    • Sample preparation methods

    • Gel concentration

  • Solution: Include positive control lysates (e.g., HeLa whole cell lysate) and molecular weight markers

• Weak or absent signal:

  • Optimize antibody concentration (typically 1:500-1:2000)

  • Increase protein loading (20-50 μg total protein)

  • Extend exposure time

  • Use enhanced chemiluminescent detection systems

  • Consider alternative antibodies targeting different epitopes

• High background:

  • Increase blocking duration (1.5 hours at room temperature with 5% non-fat milk in TBS)

  • Optimize washing (TBS-0.1% Tween, 3 times for 5 minutes each)

  • Titrate secondary antibody (typically 1:10,000)

  • Consider a more specific antibody with no reported cross-reactivity

• Subcellular fraction issues:

  • For mitochondrial TFAM, ensure proper fractionation

  • Use appropriate loading controls (α-Tubulin for cytoplasmic fractions, VDAC for mitochondrial fractions)

How can I validate the specificity of my TFAM antibody?

Comprehensive validation requires multiple approaches:

• Control experiments:

  • TFAM knockout/knockdown samples as negative controls

  • Overexpression systems as positive controls

  • Blocking peptide competition assays

• Cross-validation methods:

  • Compare staining patterns from two independent antibodies with non-overlapping epitopes

  • The Human Protein Atlas employs this method for enhanced antibody validation

• Application-specific validation:

  • For immunocytochemistry: Compare mitochondrial markers with TFAM staining patterns

  • For flow cytometry: Compare staining in known TFAM-positive and negative cell types

  • For immunohistochemistry: Assess tissue expression patterns against known TFAM distribution

• Mass spectrometry confirmation:

  • Immunoprecipitate TFAM with your antibody

  • Confirm identity through mass spectrometry analysis

How should I select a TFAM antibody when working with multiple species?

Different TFAM antibodies have varying species reactivity profiles:

• Species reactivity comparison:

  • Human-specific: Some antibodies are validated only for human samples

  • Multi-species: Others react with human, mouse, and rat samples

  • Broader reactivity: Some antibodies may cross-react with rabbit, cow, dog, guinea pig, horse, and pig samples

• Epitope conservation analysis:

  • Some epitopes vary between species. For example, the human mtTFA sequence (214-241aa EMKSWEEQMIEVGRKDLLRRTIKKQRKY) differs from mouse and rat sequences by five amino acids

  • For evolutionarily conserved regions, choose antibodies targeting these epitopes

• Validation requirements:

  • Confirm reactivity in your specific species with positive controls

  • Test multiple antibodies when working with non-standard research organisms

  • Consider custom antibody development for poorly covered species

What controls should I use to confirm TFAM antibody specificity across different applications?

Proper controls are essential for confirming specificity:

• Positive controls:

  • Cell line controls: HeLa whole cell lysate for human studies

  • Tissue controls: Brain, heart, or skeletal muscle (high TFAM expression)

• Negative controls:

  • TFAM knockout samples

  • Isotype-matched control antibodies

  • Secondary antibody-only controls

• Cross-reactivity controls:

  • Pre-adsorption with recombinant TFAM protein

  • Blocking peptide competition assays

  • Western blot validation before using in other applications

• Multi-application validation:

  • Confirm consistent results across applications (WB, IHC, ICC)

  • Document any application-specific optimizations required

How can TFAM antibodies be used to study mitochondrial dysfunction in neurodegenerative disorders?

TFAM antibodies facilitate investigation of mitochondrial abnormalities in neurodegeneration:

• Expression analysis in pathological samples:

  • Compare TFAM levels in affected vs. control tissues using immunohistochemistry

  • Quantify TFAM protein expression changes via Western blotting

  • Assess subcellular localization changes through immunofluorescence

• mtDNA maintenance assessment:

  • Combine TFAM immunostaining with mtDNA labeling techniques

  • Correlate TFAM levels with mtDNA copy number (measure using qPCR with specific primers for mtDNA genes like ND1 normalized to nuclear genes like S12)

• Nucleoid structure investigation:

  • Use super-resolution microscopy with TFAM antibodies to visualize mitochondrial nucleoid alterations

  • Combine with DNA binding proteins to assess nucleoid structural integrity

• Model system approaches:

  • Apply TFAM antibodies in cellular models of neurodegeneration

  • Use in animal models that display neurodegenerative phenotypes

  • Compare findings between models and human samples

What methodological approaches can detect TFAM post-translational modifications in stress conditions?

Detecting TFAM PTMs requires specialized techniques:

• Integrated PTM detection workflow:

  • Immunoprecipitate TFAM from stress and control conditions

  • Perform Western blot analysis with:

    • Phospho-specific antibodies

    • Acetylation-specific antibodies

    • Total TFAM antibodies

• Mass spectrometry approaches:

  • Use isotopic chemical acetylation for acetylation stoichiometry analysis

  • Apply phospho-enrichment strategies for phosphorylation site identification

  • Quantify changes in modification levels under different stressors

• Functional correlation studies:

  • Research indicates DNA-bound TFAM is less reactive to enzymatic phosphorylation and nonenzymatic acetylation

  • Assess how stress conditions affect:

    • PTM levels

    • DNA binding capacity (using EMSAs)

    • Transcription activity

• PTM interplay analysis:

  • Investigate how multiple PTMs influence each other

  • Study how combinations of modifications affect TFAM function