TSFM Antibody

What is TSFM and why is it important in research?

TSFM is a mitochondrial translation elongation factor that forms a complex with EF-Tu.GDP and facilitates the exchange of GDP to GTP. It remains bound to the aminoacyl-tRNA.EF-Tu.GTP complex until GTP hydrolysis occurs on the ribosome . TSFM plays a critical role in mitochondrial protein synthesis, and mutations in the TSFM gene are associated with combined oxidative phosphorylation deficiency-3 syndrome . Studying TSFM is important for understanding mitochondrial translation mechanisms and related pathologies.

Research significance:

• Essential component of mitochondrial protein synthesis machinery

• Located on Chromosome 12 in humans

• Implicated in rare but severe mitochondrial disorders with cardiomyopathy manifestations

• Offers insights into tissue-specific expression patterns of mitochondrial disease

What types of TSFM antibodies are available for research?

Several types of TSFM antibodies are commercially available with varying characteristics:

The choice of antibody depends on the specific application, target species, and region of interest within the TSFM protein .

How should I select the appropriate TSFM antibody for my specific research application?

Selection should be based on multiple factors:

• Experimental application: Different antibodies perform optimally in different applications. For instance, ABIN2777309 is validated only for Western Blotting, while ABIN2855337 can be used for WB, IF, IHC, and ICC .

• Species reactivity: Match the antibody's reactivity to your experimental model. For cross-species studies, consider antibodies with broader reactivity profiles like ABIN2777309, which has predicted reactivity with multiple species (Human: 100%, Mouse: 86%, Rat: 93%, etc.) .

• Epitope considerations: The target region is crucial for specific applications:

  • Middle region antibodies (ABIN2777309) may be useful for detecting specific domains

  • Full-length antibodies (ABIN2801495) might provide more robust detection of the complete protein

  • C-terminal antibodies may be better for distinguishing processed forms of the protein

• Validation evidence: Review the validation method used by the manufacturer. Cell lysate validation (as with ABIN2777309) provides evidence of detection in a complex protein mixture .

What are the optimal conditions for Western blot analysis using TSFM antibodies?

Based on available research protocols:

• Sample preparation:

  • For mitochondrial proteins like TSFM, enrichment of the mitochondrial fraction can improve detection

  • Use RIPA buffer supplemented with protease inhibitors for total protein extraction

  • Loading 20-50 μg of total protein is typically sufficient

• Electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution (TSFM is approximately 32.9 kDa)

  • Transfer to PVDF membranes at 100V for 1 hour in cold transfer buffer

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Dilute primary antibodies according to manufacturer specifications (typically 1:500 to 1:2000)

  • Incubate overnight at 4°C with gentle rocking

  • Use HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution

• Detection considerations:

  • Enhanced chemiluminescence is generally sufficient for detection

  • For low abundance samples, consider using signal enhancement systems

How can TSFM antibodies be used to study mitochondrial translation defects?

TSFM antibodies can be powerful tools for investigating mitochondrial translation defects through several methodological approaches:

• Protein level quantification: Western blot analysis using TSFM antibodies can reveal alterations in steady-state levels of TSFM, which may indicate pathological conditions. In a study of TSFM gene variants, researchers observed "dramatic steady-state level reduction of both [EF-Ts and EF-Tu] proteins in the clinically affected myocardium, which demonstrated a combined respiratory chain enzyme deficiency" .

• Co-immunoprecipitation studies: TSFM antibodies can be used to pull down TSFM-containing complexes, enabling investigation of:

  • Interactions with EF-Tu and other translation factors

  • Abnormal binding patterns in disease models

  • Post-translational modifications affecting complex formation

• Immunofluorescence for localization studies: Using antibodies like ABIN2855337 that are validated for immunofluorescence :

  • Compare TSFM distribution in healthy versus diseased tissues

  • Examine co-localization with other mitochondrial markers

  • Track changes in localization under stress conditions

• Tissue-specific expression analysis: As noted in research on TSFM mutations, there can be "compensatory response detected in patient fibroblasts [that] might explain the tissue-specific expression of TSFM-associated disease" . Immunohistochemistry with TSFM antibodies allows examination of this tissue specificity.

What approaches can be used to validate TSFM antibody specificity for research applications?

Rigorous validation is crucial for reliable research outcomes. For TSFM antibodies, consider these methodological approaches:

• Genetic knockout/knockdown controls:

  • Use CRISPR-Cas9 to generate TSFM knockout cell lines

  • Apply siRNA or shRNA to create TSFM knockdown models

  • Compare antibody signals between wild-type and knockout/knockdown samples

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide

  • Run parallel Western blots with competed and non-competed antibody

  • Specific binding should be blocked by the peptide

• Cross-species reactivity verification:

  • Test reactivity across species with known sequence homology

  • Compare observed reactivity with predicted reactivity values (e.g., Cow: 86%, Guinea Pig: 86%, Human: 100% for ABIN2777309)

• Multiple antibody concordance:

  • Use antibodies targeting different epitopes of TSFM

  • Compare detection patterns between antibodies

  • Consistent patterns across different antibodies strengthen confidence in specificity

How should researchers address inconsistent results between TSFM protein detection and gene expression data?

Discrepancies between protein and mRNA levels are common in mitochondrial research and require systematic analysis:

What are the challenges in detecting TSFM in different tissue types and how can they be overcome?

Tissue-specific detection challenges arise from several factors that require specific methodological approaches:

• Varied expression levels:

  • TSFM shows tissue-specific expression patterns

  • Heart tissue typically has high mitochondrial content and may show stronger signals

  • For tissues with lower expression, consider:

    • Signal amplification systems (TSA, polymer-based detection)

    • Longer exposure times for Western blots

    • Mitochondrial enrichment before analysis

• Background interference solutions:

  • For high-autofluorescence tissues (brain, liver):

    • Use Sudan Black B (0.1-0.3%) treatment to reduce autofluorescence

    • Consider spectral unmixing during confocal microscopy

    • Use Far-red fluorophores that compete less with autofluorescence

• Epitope masking in fixed tissues:

  • Optimize antigen retrieval for immunohistochemistry:

    • Test both heat-induced (citrate, EDTA buffers) and enzymatic methods

    • For paraffin sections, extend deparaffinization and rehydration steps

    • Consider section thickness (4-5μm optimal for most applications)

• Standardization approach:

  • Include multi-tissue Western blots to establish relative expression baselines

  • Use housekeeping proteins specific to mitochondria (e.g., VDAC) rather than global controls

  • Develop tissue-specific protocols with optimized antibody concentrations

How can modern antibody engineering techniques improve TSFM antibodies for research applications?

Recent advances in antibody technology offer several methodological improvements for TSFM research:

• Deep learning-based antibody design:

  • LSTM (Long Short Term Memory) neural networks can be employed for antibody sequence generation and prioritization

  • As demonstrated in recent research, "likelihood of generated sequences from a trained LSTM correlated well with binding affinity"

  • For TSFM research, this could enable development of antibodies with significantly higher specificity and affinity

• Microfluidics-enabled screening:

  • Novel approaches using "droplet microfluidics to encapsulate single cells into an antibody capture hydrogel" allow screening of millions of cells for antibody production

  • This technology could be applied to develop TSFM antibodies with enhanced properties:

    • Higher specificity for specific TSFM domains

    • Better species cross-reactivity

    • Improved performance in challenging applications

• Bispecific antibody applications:

  • Developing bispecific antibodies that simultaneously target TSFM and interacting partners

  • This approach could enable studies of TSFM-EF-Tu complexes in their native state

  • Similar to CD3x5T4 bispecific antibodies, which have shown "in-depth insight into the mechanism of action"

• Recombinant antibody fragments:

  • Smaller antibody fragments (Fab, scFv) can provide better tissue penetration

  • These formats may improve detection of TSFM in complex tissue samples or in vivo imaging

  • Single-domain antibodies may access epitopes not available to conventional antibodies

What new experimental approaches can researchers use to study TSFM function in mitochondrial disease models?

Cutting-edge methodologies provide new avenues for investigating TSFM's role in mitochondrial diseases:

• CRISPR-based approaches:

  • Generate precise TSFM mutations matching those found in patients

  • Create cellular models with patient-specific TSFM variants observed in mitochondrial cardiomyopathy

  • Apply CRISPRa/CRISPRi for temporal control of TSFM expression

• Proximity labeling combined with antibody detection:

  • Use BioID or APEX2 fusions with TSFM to identify proximal proteins in living cells

  • Validate interactions using co-immunoprecipitation with TSFM antibodies

  • Map the dynamic TSFM interactome under normal and stressed conditions

• Tissue-specific phenotype investigation:

  • As observed in TSFM mutation studies, there is "tissue-specific expression of TSFM-associated disease"

  • Apply single-cell techniques with TSFM antibodies to examine cell-type specific responses

  • Use organoid models to recapitulate tissue-specific TSFM functions

• Therapeutic screening platforms:

  • Develop high-throughput assays using TSFM antibodies to measure protein restoration

  • Screen small molecules that might stabilize mutant TSFM proteins

  • Evaluate compounds that enhance mitochondrial biogenesis as potential treatments

How do different TSFM antibodies compare in terms of sensitivity and specificity across applications?

Understanding performance differences is critical for selecting the optimal antibody:

Key methodological considerations:

• For critical quantitative applications, validate multiple antibodies in parallel

• Consider the target region when interpreting results (middle region vs. full-length detection)

• Species compatibility should be experimentally verified rather than relying solely on predicted reactivity

What quality control measures should be implemented when using TSFM antibodies in long-term research projects?

To ensure consistent results across extended research timelines:

• Antibody lot testing protocol:

  • Test each new lot against a standard sample

  • Maintain reference blots/images for comparison

  • Document lot-to-lot variation in sensitivity and background

• Storage and handling optimization:

  • Aliquot antibodies upon receipt to minimize freeze-thaw cycles

  • For TSFM antibodies, stability data indicates: "Store at 4°C if entire vial will be used within 2-4 weeks. Store frozen at -20°C for longer periods"

  • Add carrier protein (0.1% HSA or BSA) for long-term storage

• Internal standard implementation:

  • Include positive control samples in each experiment

  • Consider using recombinant TSFM protein (like the one described in source ) as a standard

  • Develop a quantitative Western blot protocol with standard curves

• Documentation and standardization:

  • Maintain detailed records of all experimental parameters

  • Create standard operating procedures for key techniques

  • Document antibody performance metrics for reproducibility

By implementing these measures, researchers can minimize variability and ensure reliable data across multiple experiments and extended research timelines.