TOMM70 Antibody

What is TOMM70 and why is it significant in mitochondrial research?

TOMM70 (Translocase of Outer Mitochondrial Membrane 70) functions as a receptor within the preprotein translocase complex of the outer mitochondrial membrane (TOM complex). It serves as a docking partner for cytosolic chaperone proteins and participates in the uptake of newly synthesized chaperone-bound proteins during mitochondrial biogenesis . TOMM70 recognizes and mediates the translocation of mitochondrial preproteins from the cytosol into the mitochondria in a chaperone-dependent manner . Its significance extends beyond protein import, as it also plays roles in Parkinson's disease pathology through PINK1/Parkin recruitment and in antiviral immune responses .

What applications can TOMM70 antibodies be utilized for in laboratory research?

TOMM70 antibodies demonstrate versatility across multiple experimental applications as shown in the following table:

The selection of application should be guided by experimental requirements, with appropriate optimization for each specific antibody clone .

How should TOMM70 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of TOMM70 antibodies is crucial for maintaining their effectiveness:

• Store antibodies at -20°C for long-term storage (stable for approximately one year after shipment)

• For short-term storage, antibodies can be kept at 4°C for up to three months

• Avoid repeated freeze-thaw cycles as this significantly reduces antibody performance

• Do not aliquot certain antibody formulations (specifically noted on product documentation)

• Most TOMM70 antibodies are supplied in PBS containing 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations may contain BSA as a stabilizer, especially in smaller volumes

Following these guidelines ensures antibody integrity and consistent experimental results over time .

How do the interactomes of TOMM20 and TOMM70 differ, and what does this reveal about their functional specialization?

Proximity labeling experiments using APEX2 technology have revealed distinct interactome patterns for TOMM20 and TOMM70, reflecting their specialized functions:

• TOMM20-APEX2 interactome identified 21 mitochondrial outer membrane (MOM) proteins, while TOMM70-APEX2 identified only 7 MOM proteins

• TOMM20 appears to interact with more MOM proteins compared to TOMM70, likely due to its more stable association with the TOM complex

• TOMM70 was present in the dataset of potential interactors of TOMM20-APEX2, but interestingly, TOMM20 was not prominently identified in TOMM70-APEX2 datasets

• TOMM20-APEX2 interactome revealed enrichment for RNA-binding proteins (RBPs), including SYNJ2BP, PAIP1, MAVS, and PABPC4L, which were not enriched in the TOMM70-APEX2 interactome

• Comparison with matrix-targeted APEX2 (Mito-APEX2) confirmed that both TOMM20 and TOMM70 primarily interact with proteins at the MOM-cytoplasm interface

These differences suggest that TOMM20 and TOMM70 have distinct functional roles within the TOM complex, with TOMM70 potentially more specialized for chaperone-mediated protein import .

What technical considerations should researchers address when validating new TOMM70 antibodies?

When validating TOMM70 antibodies for research applications, multiple technical considerations should be addressed:

• Specificity verification:

  • Western blot analysis should confirm a single band at approximately 75 kDa in relevant cell and tissue lysates

  • Testing across multiple cell lines (e.g., HeLa, HepG2, MCF-7, HEK-293, Jurkat) confirms cross-reactivity

  • Knockout/knockdown controls are essential to confirm specificity

• Cross-species reactivity assessment:

  • Documented reactivity with human, mouse, and rat samples should be verified in your specific models

  • Species differences in protein expression levels should be considered when interpreting results

• Subcellular localization confirmation:

  • Immunofluorescence should show characteristic mitochondrial staining patterns

  • Co-localization with established mitochondrial markers provides additional validation

  • Subcellular fractionation can verify enrichment in mitochondrial versus cytosolic fractions

• Application-specific optimization:

  • Each application requires individual optimization of antibody concentration

  • Antigen retrieval methods for IHC may vary (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Sample-dependent variability necessitates titration in each testing system

These validation steps ensure reliable and reproducible results in experimental applications .

How can proximity labeling techniques be implemented to study the TOMM70 interactome?

Proximity labeling using engineered peroxidases like APEX2 provides a powerful approach for studying TOMM70 interactomes:

• Construct design and validation:

  • Generate fusion proteins linking TOMM70 to APEX2

  • Validate correct targeting using subcellular fractionation and co-localization with endogenous TOMM70

  • Confirm similar expression levels between fusion proteins and endogenous counterparts

• Experimental setup:

  • Use doxycycline-inducible expression systems for controlled expression of TOMM70-APEX2

  • Include appropriate controls: uninduced cells, cytosolic APEX2 (NES-APEX2), and matrix-targeted APEX2 (Mito-APEX2)

  • Incubate cells with biotin-phenol followed by brief H₂O₂ treatment to activate APEX2-mediated biotinylation

• Protein capture and analysis:

  • Isolate biotinylated proteins using streptavidin beads after cell lysis

  • Perform on-bead tryptic digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS)

  • Apply label-free quantification (LFQ) for data processing and imputation of missing values to increase identifications

• Data interpretation:

  • Compare TOMM70-APEX2 datasets with controls to identify significantly enriched interactors

  • Apply statistical thresholds (p ≤ 0.05 for significant, p ≤ 0.01 for stringently significant)

  • Analyze mitochondrial and non-mitochondrial interactors to understand TOMM70's roles at the mitochondria-cytosol interface

This approach has successfully identified distinct proteins in the TOMM70 interactome, revealing its unique functional associations compared to other TOM complex components .

What is the role of TOMM70 in innate immune signaling, and how can this be experimentally investigated?

TOMM70 plays a critical role in innate immune signaling, particularly in antiviral responses:

• TOMM70's role in antiviral signaling:

  • TOMM70 interacts with mitochondrial antiviral-signaling protein (MAVS) upon RNA virus infection

  • It mediates the activation of TBK1 and IRF3, key components of antiviral signaling

  • The clamp domain (R192) of TOMM70 interacts with the C-terminal motif (EEVD) of Hsp90, recruiting TBK1/IRF3 to mitochondria

  • Disruption of this interaction impairs activation of TBK1 and IRF3

  • During Sendai virus infection, TOMM70 recruits HSP90AA1:IRF3:BAX complexes to mitochondria, inducing apoptosis

• Experimental approaches to investigate this function:

  • Protein interaction studies:

    • Co-immunoprecipitation using anti-TOMM70 antibodies to identify virus-induced interactions

    • Proximity ligation assays to visualize TOMM70-MAVS-HSP90 interactions in situ

  • Functional assessment:

    • TOMM70 knockdown/knockout followed by viral challenge to assess effects on IRF3 activation and interferon production

    • Site-directed mutagenesis of the clamp domain (R192) to disrupt HSP90 interaction

    • Subcellular fractionation to analyze recruitment of signaling components to mitochondria

  • Imaging approaches:

    • Immunofluorescence co-localization of TOMM70 with MAVS, TBK1, and IRF3 during viral infection

    • Live-cell imaging using fluorescently tagged proteins to track dynamics of complex formation

• Relevance to viral infections:

  • SARS-CoV-2 Orf9b protein binds to TOMM70, potentially suppressing interferon responses

  • This interaction may represent a viral immune evasion strategy that can be targeted therapeutically

These experimental approaches allow investigation of TOMM70's dual roles in mitochondrial protein import and innate immune signaling .

How can researchers effectively distinguish between TOMM70's roles in protein import versus innate immunity in their experimental designs?

Distinguishing between TOMM70's dual functions requires carefully designed experimental approaches:

• Domain-specific mutations:

  • Generate constructs with mutations in specific functional domains:

    • TPR (tetratricopeptide repeat) domains critical for chaperone interactions in protein import

    • The clamp domain (R192) essential for HSP90-mediated immune signaling

  • These allow separation of effects on protein import from those on immune signaling

• Temporal analysis:

  • Establish time-course experiments following stimuli:

    • Mitochondrial stress (e.g., CCCP treatment) activates protein import functions

    • Viral infection or poly(I:C) treatment triggers immune signaling roles

  • Analyze TOMM70 interactions at different timepoints using immunoprecipitation with anti-TOMM70 antibodies

• Compartment-specific analysis:

  • Employ subcellular fractionation to isolate:

    • TOMM70 associated with intact mitochondrial membranes (protein import)

    • TOMM70 in complexes with cytosolic signaling proteins (immune function)

  • Western blotting with anti-TOMM70 antibodies can detect distribution between compartments

• Interactome comparison:

  • Compare TOMM70 interactors under different conditions:

    • Basal conditions (primarily protein import)

    • Viral challenge (immune signaling activation)

  • Mass spectrometry analysis of TOMM70 immunoprecipitates can reveal condition-specific interactors

• Selective targeting:

  • RNA interference targeting specific TOMM70-interacting partners:

    • Chaperones involved in protein import

    • Components of immune signaling pathways (MAVS, TBK1, IRF3)

  • Monitor effects on both functions using appropriate readouts

This experimental strategy enables researchers to parse the multifunctional nature of TOMM70 and determine how these functions may be interconnected or independently regulated .

What are common technical challenges when using TOMM70 antibodies, and how can these be addressed?

Researchers frequently encounter several technical challenges when working with TOMM70 antibodies:

• Background signal in Western blotting:

  • Optimize antibody dilution within the recommended range (1:1000 to 1:50000)

  • Increase blocking stringency (5% non-fat dry milk or BSA in TBST)

  • Use freshly prepared buffers and increase washing duration/frequency

  • For mouse tissues with high fat content, extend blocking time and use additional detergents

• Weak or absent signal:

  • Verify protein loading (75 kDa band)

  • Ensure sample preparation preserves mitochondrial proteins (avoid harsh detergents initially)

  • Check antibody storage conditions and expiration

  • For specific cell types with low expression, increase protein loading or use more sensitive detection methods

• Non-specific bands:

  • Validate antibody specificity using knockout/knockdown controls

  • Test multiple antibody clones if available

  • Optimize SDS-PAGE conditions (10% gels typically work well for 75 kDa TOMM70)

  • Increase antibody specificity with longer primary antibody incubation at 4°C overnight

• Immunofluorescence optimization:

  • Paraformaldehyde fixation (4%) works well for preserving mitochondrial structure

  • Test a range of antibody dilutions (1:50 to 1:500)

  • Co-stain with established mitochondrial markers to confirm localization

  • Optimize permeabilization conditions (0.1-0.5% Triton X-100) if penetration is an issue

• Immunohistochemistry challenges:

  • Test different antigen retrieval methods (TE buffer pH 9.0 often works better than citrate buffer pH 6.0)

  • Use positive control tissues with known TOMM70 expression (brain tissue works well)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Block endogenous peroxidase activity thoroughly before antibody application

Addressing these common challenges through systematic optimization will significantly improve experimental outcomes with TOMM70 antibodies .

How can researchers ensure reproducibility across different TOMM70 antibody sources in comparative studies?

Ensuring reproducibility when using TOMM70 antibodies from different sources requires systematic validation and standardization:

• Initial antibody characterization:

  • Perform side-by-side Western blots with multiple antibodies using the same samples and conditions

  • Document specifics for each antibody: host species, clonality, immunogen, and epitope location

  • Validate recognition of recombinant versus endogenous TOMM70

• Standardization protocol:

  • Establish a standardized protocol for each application with detailed parameters:

    • Sample preparation method (lysis buffer composition, protein concentration)

    • Blocking conditions (agent, concentration, duration)

    • Antibody dilution ranges optimized for each source

    • Incubation times and temperatures

    • Detection methods and exposure settings

• Cross-validation strategies:

  • Employ multiple detection methods (e.g., Western blot, IF, IHC) with each antibody

  • Use genetic controls (siRNA knockdown or CRISPR knockout) to verify specificity of each antibody

  • Document batch-to-batch variation within the same supplier

  • Create internal reference samples to be used across all experiments

• Application-specific considerations:

  • For Western blotting: standardize protein loading, transfer conditions, and detection method

  • For immunofluorescence: use consistent fixation and permeabilization protocols

  • For IHC: standardize tissue processing, antigen retrieval, and counterstaining

• Documentation and reporting:

  • Maintain detailed records of antibody performance characteristics

  • Record catalog numbers, lot numbers, and dates of experiments

  • Document any deviations from standard protocols and their effects

  • Report comprehensive antibody information in publications following the antibody reporting guidelines

This systematic approach minimizes variability when using different TOMM70 antibody sources, ensuring more reliable and reproducible research outcomes .

How can TOMM70 antibodies be utilized to investigate the role of TOMM70 in SARS-CoV-2 infection mechanisms?

TOMM70 antibodies offer valuable tools for investigating the interaction between SARS-CoV-2 and mitochondrial biology:

• Investigating viral protein-TOMM70 interactions:

  • Use anti-TOMM70 antibodies for co-immunoprecipitation experiments to capture SARS-CoV-2 Orf9b-TOMM70 complexes

  • Perform immunofluorescence co-localization studies of TOMM70 and viral proteins in infected cells

  • Develop proximity ligation assays (PLA) using anti-TOMM70 antibodies paired with anti-Orf9b antibodies to visualize interactions in situ

• Analyzing alterations in TOMM70 expression and localization:

  • Monitor TOMM70 expression levels before and after SARS-CoV-2 infection using Western blotting

  • Track subcellular redistribution of TOMM70 during infection using fractionation followed by immunoblotting

  • Perform time-course immunofluorescence studies with anti-TOMM70 antibodies during viral infection

• Assessing impact on interferon signaling:

  • Analyze TOMM70-TBK1-IRF3 complex formation with and without viral infection using immunoprecipitation

  • Determine how Orf9b binding affects TOMM70's ability to recruit HSP90:IRF3 using pull-down assays

  • Compare interferon response gene expression in cells with normal versus disrupted TOMM70-HSP90 interaction

• Therapeutic targeting approaches:

  • Screen for compounds that may disrupt Orf9b-TOMM70 interaction using competitive binding assays

  • Investigate whether antibodies that recognize specific TOMM70 domains can block viral protein binding

  • Develop cell-permeable peptides that mimic TOMM70-binding regions to competitively inhibit viral protein interactions

• Structural studies:

  • Use antibody epitope mapping to identify critical regions of TOMM70 involved in viral protein binding

  • Perform immunoprecipitation with anti-TOMM70 antibodies followed by mass spectrometry to identify post-translational modifications induced by viral infection

These approaches leverage TOMM70 antibodies to elucidate how SARS-CoV-2 manipulates mitochondrial function through TOMM70 interaction, potentially revealing new therapeutic targets .

What methodologies can be employed to study the relationship between TOMM70 dysfunction and neurodegenerative diseases?

TOMM70's involvement in protein import and quality control processes suggests potential roles in neurodegenerative diseases that can be investigated using several methodologies:

• Patient-derived tissue analysis:

  • Compare TOMM70 expression and localization in post-mortem brain tissue from Parkinson's disease patients versus controls using immunohistochemistry

  • Perform Western blot analysis with anti-TOMM70 antibodies on brain region-specific lysates to quantify expression differences

  • Examine co-localization of TOMM70 with PINK1/Parkin in disease versus healthy tissue using dual immunofluorescence

• Cell models of neurodegeneration:

  • Establish neuronal cell lines with TOMM70 knockdown/knockout to assess effects on mitochondrial function

  • Use anti-TOMM70 antibodies to monitor TOMM70 recruitment to depolarized mitochondria following CCCP treatment

  • Track TOMM70-PINK1-Parkin interactions during mitophagy using immunoprecipitation and Western blotting

• Protein aggregation studies:

  • Investigate TOMM70 interaction with disease-associated proteins (α-synuclein, tau, huntingtin) using co-immunoprecipitation

  • Analyze whether protein aggregates sequester TOMM70 away from mitochondria using subcellular fractionation

  • Perform proximity ligation assays between TOMM70 and neurodegeneration-associated proteins

• Animal model approaches:

  • Generate conditional TOMM70 knockout in specific neuronal populations to assess neurodegenerative phenotypes

  • Perform immunohistochemistry with anti-TOMM70 antibodies in transgenic models of Parkinson's, Alzheimer's, or other neurodegenerative diseases

  • Assess rescue of phenotypes through TOMM70 modulation in disease models

• Mitochondrial import assays:

  • Develop in vitro assays using isolated mitochondria to assess TOMM70-dependent protein import efficiency

  • Compare import capacity between mitochondria from healthy versus diseased states

  • Use antibody inhibition approaches to determine the consequences of impaired TOMM70 function on import of specific substrates

These methodologies provide complementary approaches to understand how TOMM70 dysfunction may contribute to neurodegenerative disease pathogenesis, potentially revealing new therapeutic targets .