TIM17-1 Antibody

Basic Research Questions

How do I validate the specificity of TIM17-1 antibodies in plant mitochondrial studies?

To validate specificity:

• Perform knockout controls: Use AtTim17-1 mutant lines (e.g., T-DNA insertions at −402 bp or +360 bp) and compare protein levels via immunoblotting. A 60–70% transcript reduction in mutants should correlate with diminished antibody signal .

• Cross-reactivity tests: Validate using isoforms (e.g., AtTim17-2 or AtTim17-3). Antibodies targeting AtTim17-1 may cross-react with AtTim17-2 due to shared epitopes, but isoforms can be distinguished by molecular weight (32 kDa vs. 29 kDa) .

• Recombinant protein validation: Express recombinant Tim17-1 and Tim17-2 to confirm antibody binding specificity .

What protocols are recommended for detecting TIM17-1 in mammalian cell lines?

• Western blot: Use 10–20 µg mitochondrial lysate, 12% SDS-PAGE, and antibodies like ab192246 (predicted 18 kDa, observed 17 kDa) .

• Immunofluorescence: Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and use secondary antibodies (e.g., Alexa Fluor® 555) for mitochondrial co-localization .

• Immunoprecipitation: Employ digitonin-solubilized mitochondria and Ni-agarose beads for Tim17-1 complex isolation .

Advanced Research Questions

How can TIM17-1 antibody resolve isoforms in organisms with paralogs (e.g., Arabidopsis or human)?

What functional assays link TIM17-1 to mitochondrial DNA (mtDNA) stability?

• Yeast suppression assays: Overexpress TIM17 in ilv5Δ or abf2Δ mutants to assess mtDNA retention (76% suppression in ilv5Δ; 32% in abf2Δ) .

• Human cybrid models: Introduce TIM17A into NT2 cells with A3243G mutant mtDNA. Overexpression stabilizes mtDNA levels (13–17-fold increase vs. empty vector) .

• QRT-PCR validation: Monitor mtDNA copy number using ND1 (mtDNA) vs. 18S rRNA (nuclear DNA) primers .

How to troubleshoot cross-reactivity with TIM17-2 in co-immunoprecipitation experiments?

• Epitope mapping: Use truncated Tim17-1/2 constructs to identify antibody-binding regions .

• Competition assays: Pre-incubate antibodies with recombinant Tim17-2 to block non-specific binding .

• Subcellular fractionation: Isolate mitochondria rigorously to reduce cytosolic contamination .

Methodological Considerations

What structural motifs in Tim17-1 are critical for mitochondrial import?

• Transmembrane domains (TMs): TM1 and TM4 are essential for proper localization (Fig. 1A). Deletion of TM1 (ΔN50) or TM4 (ΔC31) disrupts mitochondrial integration .

• C-terminal ITS: Residues 121–152 in T. brucei Tim17 contain an internal targeting signal (ITS) sufficient for GFP import .

How to design experiments analyzing Tim17-1’s role in presequence protein translocation?

• Crosslinking with preproteins: Use cysteine-rich Tim17-1 mutants and bifunctional crosslinkers (e.g., BMH) to trap transient interactions .

• Electrophysiology: Incorporate Tim17-1 into proteoliposomes to measure channel activity in response to presequence peptides .

• Charge-swap mutagenesis: Replace conserved negative charges (e.g., Glu94) to disrupt presequence binding .

Data Contradiction Analysis

Why do some studies report varying molecular weights for Tim17-1?

How to reconcile conflicting results on Tim17-1’s role in mtDNA stability?

• Model organism differences: Yeast TIM17 suppresses mtDNA loss broadly, while human TIM17A specifically stabilizes A3243G mtDNA .

• Experimental variables: Strain background (e.g., ilv5Δ vs. abf2Δ) and growth conditions (fermentable vs. non-fermentable carbon sources) alter outcomes .